Cell

ABSTRACT

The present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising: (i) an antigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and (iv) an endodomain wherein the antigen binding domains of the first and second CARs bind to different antigens, and wherein each of the first and second CARs is an activating CAR comprising an activating endodomain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a Continuation of U.S. application Ser. No. 15/037,391, filedMay 18, 2016, which is a U.S. National Phase of InternationalApplication No. PCT/GB2014/053451, filed Nov. 21, 2014, which claimspriority to Great Britain Application No. 1410934.2, filed Jun. 19, 2014and Great Britain Application No. 1320573.7, filed Nov. 21, 2013.

FIELD OF THE INVENTION

The present invention relates to a cell which comprises more than onechimeric antigen receptor (CAR). The cell may be capable of specificallyrecognising a target cell, due to a differential pattern of expression(or non-expression) of two or more antigens by the target cell.

BACKGROUND TO THE INVENTION

A number of immunotherapeutic agents have been described for use incancer treatment, including therapeutic monoclonal antibodies (mAbs),immunoconjugated mAbs, radioconjugated mAbs and bi-specific T-cellengagers.

Typically these immunotherapeutic agents target a single antigen: forinstance, Rituximab targets CD20; Myelotarg targets CD33; andAlemtuzumab targets CD52.

However, it is relatively rare for the presence (or absence) of a singleantigen effectively to describe a cancer.

A particular problem in the field of oncology is provided by theGoldie-Coldman hypothesis: which describes that the sole targeting of asingle antigen may result in tumour escape by modulation of said antigendue to the high mutation rate inherent in most cancers. This modulationof antigen expression may reduce the efficacy of knownimmunotherapeutics.

Tumour heterogeneity describes the observation that different tumourcells can show distinct morphological and phenotypic profiles, includingcellular morphology, gene expression, metabolism, proliferation andmetastatic potential. This phenomenon occurs both between tumours andwithin tumours. Tumour heterogeneity has been observed in leukaemias,breast, prostate, colon, brain, head and neck, bladder and gynecologicalcancers, for example. Tumour heterogeneity may result in the expressionof different antigens on the surface of cells within a tumour or betweentumours. This heterogeneity of cancer cells introduces significantchallenges in designing effective treatment strategies.

There is thus a need for immunotherapeutic agents which are capable ofmore targeting to reflect the complex pattern of marker expression thatis associated with many cancers.

Chimeric Antigen Receptors (CARs)

Chimeric antigen receptors are proteins which graft the specificity of amonoclonal antibody (mAb) to the effector function of a T-cell. Theirusual form is that of a type I transmembrane domain protein with anantigen recognizing amino terminus, a spacer, a transmembrane domain allconnected to a compound endodomain which transmits T-cell survival andactivation signals (see FIG. 1A).

The most common form of these molecules are fusions of single-chainvariable fragments (scFv) derived from monoclonal antibodies whichrecognize a target antigen, fused via a spacer and a trans-membranedomain to a signaling endodomain. Such molecules result in activation ofthe T-cell in response to recognition by the scFv of its target. When Tcells express such a CAR, they recognize and kill target cells thatexpress the target antigen. Several CARs have been developed againsttumour associated antigens, and adoptive transfer approaches using suchCAR-expressing T cells are currently in clinical trial for the treatmentof various cancers.

It has been observed that using a CAR approach for cancer treatment,tumour heterogeneity and immunoediting can cause escape from CARtreatment. For example, in the study described by Grupp et al (2013; NewEng. J. Med 368:1509-1518) CAR-modified T cell approach was used for thetreatment of acute lymphoid leukemia. It was found that one patient ofthe anti-CD19 CAR trial relapsed with CD19-negative disease two monthsafter treatment.

There is thus a need for alternative CAR treatment approaches whichaddress the problems of cancer escape and tumour heterogeneity.

Expression of Two CAR Binding Specificities

Bispecific CARs known as tandem CARs or TanCARs have been developed inan attempt to target multiple cancer specific markers simultaneously. Ina TanCAR, the extracellular domain comprises two antigen bindingspecificities in tandem, joined by a linker. The two bindingspecificities (scFvs) are thus both linked to a single transmembraneportion: one scFv being juxtaposed to the membrane and the other beingin a distal position.

Grada et al (2013, Mol Ther Nucleic Acids 2:e105 describes a TanCARwhich includes a CD19-specific scFv, followed by a Gly-Ser linker andthen a HER2-specific scFv. The HER2-scFv was in the juxta-membraneposition, and the CD19-scFv in the distal position. The Tan CAR wasshown to induce distinct T cell reactivity against each of the twotumour restricted antigens. This arrangement was chosen because therespective lengths of HER2 (632 aa/125 Å) and CD19 (280aa, 65 Å) lendsitself to that particular spatial arrangement. It was also known thatthe HER2 scFv bound the distal-most 4 loops of HER2.

The problem with this approach is that the juxta-membrane scFv may beinaccessible due to the presence of the distal scFv, especially which itis bound to the antigen. In view of the need to choose the relativepositions of the two scFvs in view of the spatial arrangement of theantigen on the target cell, it may not be possible to use this approachfor all scFv binding pairs. Moreover, it is unlikely that the TanCarapproach could be used for more than two scFvs, a TanCAR with three ormore scFvs would be a very large molecule and the scFvs may well foldback on each other, obscuring the antigen-binding sites. It is alsodoubtful that antigen-binding by the most distal scFv, which isseparated from the transmembrane domain by two or more further scFvs,would be capable of triggering T cell activation.

There is thus a need for an alternative approach to express two CARbinding specificities on the surface of a cell such as a T cell.

DESCRIPTION OF THE FIGURES

FIG. 1A-1D: FIG. 1A. Generalized architecture of a CAR: A binding domainrecognizes antigen; the spacer elevates the binding domain from the cellsurface; the trans-membrane domain anchors the protein to the membraneand the endodomain transmits signals. FIG. 1B-1D. Different generationsand permutations of CAR endodomains: FIG. 1B. initial designstransmitted ITAM signals alone through FcεR1-γ or CD3ζ endodomain, whilelater designs transmitted additional FIG. 1C. one or FIG. 1D. twoco-stimulatory signals in cis.

FIG. 2: Schematic diagram illustrating the invention

The invention relates to engineering T-cells to respond to logical rulesof target cell antigen expression. This is best illustrated with animaginary FACS scatter-plot. Target cell populations express both,either or neither of antigens “A” and “B”. Different target populations(marked in red) are killed by T-cells transduced with a pair of CARsconnected by different gates. With OR gated receptors, bothsingle-positive and double-positive cells will be killed. With AND gatedreceptors, only double-positive target cells are killed. With AND NOTgating, double-positive targets are preserved while single-positivetargets

FIG. 3: Creation of target cell populations

SupT1 cells were used as target cells. These cells were transduced toexpress either CD19, CD33 or both CD19 and CD33. Target cells werestained with appropriate antibodies and analysed by flow cytometry.

FIG. 4: Cassette design for an OR gate

A single open reading frame provides both CARs with an in-frame FMD-2Asequence resulting in two proteins. Signal1 is a signal peptide derivedfrom IgG1 (but can be any effective signal peptide). scFv1 is thesingle-chain variable segment which recognizes CD19 (but can be a scFvor peptide loop or ligand or in fact any domain which recognizes anydesired arbitrary target). STK is the CD8 stalk but may be any suitableextracellular domain. CD28tm is the

CD28 trans-membrane domain but can by any stable type I proteintransmembrane domain and CD3Z is the CD3 Zeta endodomain but can be anyendodomain which contains ITAMs. Signal2 is a signal peptide derivedfrom CD8 but can be any effective signal peptide which is different inDNA sequence from signal1. scFv recognizes CD33 but as for scFv1 isarbitrary. HC2CH3 is the hinge-CH2-CH3 of human IgG1 but can be anyextracellular domain which does not cross-pair with the spacer used inthe first CAR. CD28tm′ and CD3Z′ code for the same protein sequence asCD28tm and CD3Z but are codon-wobbled to prevent homologousrecombination.

FIG. 5A-5B: Schematic representation of the chimeric antigen receptors(CARs) for an OR gate

Stimulatory CARs were constructed consisting of either an N-terminalFIG. 5A. anti-CD19 scFv domain followed by the extracellular hingeregion of human CD8 or FIG. 5B. anti-CD33 scFv domain followed by theextracellular hinge, CH2 and CH3 (containing a pvaa mutation to reduceFcR binding) region of human IgG1. Both receptors contain a human CD28transmembrane domain and a human CD3 Zeta (CD247) intracellular domain.“S” depicts the presence of disulphide bonds.

FIG. 6: Expression data showing co-expression of both CARs on thesurface of one T-cell.

FIG. 7: Functional analysis of the OR gate

Effector cells (5×10{circumflex over ( )}4 cells) expressing the OR gateconstruct were co-incubated with a varying number of target cells andIL-2 was analysed after 16 hours by ELISA. The graph displays theaverage maximum IL-2 secretion from a chemical stimulation (PMA andlonomycin) of the effector cells alone and the average background IL-2from effector cells without any stimulus from three replicates.

FIG. 8: Cartoon showing both versions of the cassette used to expressboth AND gates Activating and inhibiting CARs were co-expressed onceagain using a FMD-2A sequence. Signal1 is a signal peptide derived fromIgG1 (but can be any effective signal peptide). scFv1 is thesingle-chain variable segment which recognizes CD19 (but can be a scFvor peptide loop or ligand or in fact any domain which recognizes anydesired arbitrary target). STK is the CD8 stalk but may be any non-bulkyextracellular domain. CD28tm is the CD28 trans-membrane domain but canby any stable type I protein transmembrane domain and CD3Z is the CD3Zeta endodomain but can be any endodomain which contains ITAMs. Signal2is a signal peptide derived from CD8 but can be any effective signalpeptide which is different in DNA sequence from signal1. scFv recognizesCD33 but as for scFv1 is arbitrary. HC2CH3 is the hinge-CH2-CH3 of humanIgG1 but can be any bulky extracellular domain. CD45 and CD148 are thetransmembrane and endodomains of CD45 and CD148 respectively but can bederived from any of this class of protein.

FIG. 9: Schematic representation of the protein structure of chimericantigen receptors (CARs) for the AND gates

The stimulatory CAR consisting of an N-terminal anti-CD19 scFv domainfollowed by the extracellular stalk region of human CD8, human CD28transmembrane domain and human CD3 Zeta (CD247) intracellular domain.Two inhibitory CARs were tested. These consist of an N-terminalanti-CD33 scFv domain followed by the extracellular hinge, CH2 and CH3(containing a pvaa mutation to reduce FcR binding) region of human IgG1followed by the transmembrane and intracellular domain of either humanCD148 or CD45. “S” depicts the presence of disulphide bonds.

FIG. 10: Co-expression of activation and inhibitory CARs

BW5147 cells were used as effector cells and were transduced to expressboth the activation anti-CD19 CAR and one of the inhibitory anti-CD33CARs. Effector cells were stained with CD19-mouse-Fc and CD33-rabbit-Fcand with appropriate secondary antibodies and analysed by flowcytometry.

FIG. 11A-11B: Functional analysis of the AND gates

Effector cells (5×10{circumflex over ( )}4 cells) expressing activationanti-CD19 CAR and the inhibitory anti-CD33 CAR with the FIG. 11A. CD148or FIG. 11B. CXD45 intracellular domain were co-incubated with a varyingnumber of target cells and IL-2 was analysed after 16 hours by ELISA.The graph displays the maximum IL-2 secretion from a chemicalstimulation (PMA and lonomycin) of the effector cells alone and thebackground IL-2 from effector cells without any stimulus from threereplicates.

FIG. 12: Cartoon showing three versions of the cassette used to generatethe AND NOT gate

Activating and inhibiting CARs were co-expressed once again using aFMD-2A sequence. Signal1 is a signal peptide derived from IgG1 (but canbe any effective signal peptide). scFv1 is the single-chain variablesegment which recognizes CD19 (but can be a scFv or peptide loop orligand or in fact any domain which recognizes any desired arbitrarytarget). STK is the human CD8 stalk but may be any non-bulkyextracellular domain. CD28tm is the CD28 trans-membrane domain but canby any stable type I protein transmembrane domain and CD3Z is the CD3Zeta endodomain but can be any endodomain which contains ITAMs. Signal2is a signal peptide derived from CD8 but can be any effective signalpeptide which is different in DNA sequence from signal1. scFv recognizesCD33 but as for scFv1 is arbitrary. muSTK is the mouse CD8 stalk but canbe any spacer which co-localises but does not cross-pair with that ofthe activating CAR. dPTPN6 is the phosphatase domain of PTPN6. LAIR1 isthe transmembrane and endodomain of LAIR1. 2Aw is a codon-wobbledversion of the FMD-2A sequence. SH2-CD148 is the SH2 domain of PTPN6fused with the phosphatase domain of CD148.

FIG. 13A-13D: Schematic representation of the chimeric antigen receptors(CARs) for the NOT AND gates

FIG. 13A. A stimulatory CAR consisting of an N-terminal anti-CD19 scFvdomain followed by the stalk region of human CD8, human CD28transmembrane domain and human CD247 intracellular domain. FIG. 13B. Aninhibitory CAR consisting of an N-terminal anti-CD33 scFv domainfollowed by the stalk region of mouse CD8, transmembrane region of mouseCD8 and the phosphatase domain of PTPN6. FIG. 13C. an inhibitory CARconsisting of an N-terminal anti-CD33 scFv domain followed by the stalkregion of mouse CD8 and the transmembrane and intracellular segments ofLAIR1. FIG. 13D. An inhibitory CAR identical to previous CAR except itis co-expressed with a fusion protein of the PTPN6 SH2 domain and theCD148 phosphatase domain.

FIG. 14A-14B: Functional analysis of the NOT AND gate

Effector cells (5×10{circumflex over ( )}4 cells) expressing the FIG.14A. full length SHP-1 or FIG. 14B. truncated form of SHP-1 wereco-incubated with a varying number of target cells and IL-2 was analysedafter 16 hours by ELISA. The graph displays the average maximum IL-2secretion from a chemical stimulation (PMA and lonomycin) of theeffector cells alone and the average background IL-2 from effector cellswithout any stimulus from three replicates.

FIG. 15: Amino acid sequence of an OR gate

FIG. 16: Amino acid sequence of a CD148 and a CD45 based AND gate

FIG. 17: Amino acid sequence of three AND NOT gates

FIG. 18A-18C: Dissection of AND gate function

FIG. 18A. The prototype AND gate is illustrated on the right and itsfunction in response to CD19, CD33 single and CD19, CD33 double positivetargets is shown on the left. FIG. 18B. The scFvs are swapped so theactivating endodomain is triggered by CD33 and the inhibitory endodomainis activated by CD19. This AND gate remains functional despite this scFvswap. FIG. 18C. The CD8 mouse stalk replaced Fc in the spacer of theinhibitory CAR. With this modification, the gate fails to respond toeither CD19 single positive or CD19, CD33 double positive targets.

FIG. 19A-19D: Expression of target antigens on artificial target cells

FIG. 19A. Shows flow cytometry scatter plots CD19 vs CD33 of theoriginal set of artificial target cells derived from SupT1 cells. Fromleft to right: double negative SupT1 cells, SupT1 cells positive forCD19, positive for CD33 and positive for both CD19 and CD33. FIG. 19B.Shows flow cytometry scatter plots CD19 vs GD2 of the artificial targetcells generated to test the CD19 AND GD2 gate: From left to right:negative SupT1 cells, SupT1 cells expressing CD19, SupT1 cellstransduced with GD2 and GM3 synthase vectors which become GD2 positiveand SupT1 cells transduced with CD19 as well as GD2 and GM3 synthasewhich are positive for both GD2 and CD19. FIG. 19C. Shows flow cytometryscatter plots of CD19 vs EGFRvIII of the artificial targets generated totest the CD19 AND EGFRvIII gate. From left to right: negative SupT1cells, SupT1 cells expressing CD19, SupT1 cells transduced with EGFRvIIIand SupT1 cells transduced with both CD19 and EGFRvIII. FIG. 19D. Showsflow cytometry scatter plots of CD19 vs CD5 of the artificial targetsgenerated to test the CD19 AND CD5 gate. From left to right: negative293T cells, 293T cells transduced with CD19, 293T cells transduced withCD5, 293T cells transduced with both CD5 and CD19 vectors.

FIG. 20A-20D: Generalizability of the AND gate

FIG. 20A. Cartoon of AND gate modified so the second CAR's specificityis changed from the original specificity of CD33, to generate 3 newCARs: CD19 AND GD2, CD19 AND EGFRvIII, CD19 AND CD5. FIG. 20B. CD19 ANDGD2 AND gate: Left: expression of AND gate is shown recombinant CD19-Fcstaining (x-axis) for the CD19 CAR, versus anti-human-Fc staining(Y-axis) for the GD2 CAR. Right: function in response to single positiveand double positive targets. FIG. 20C. CD19 AND EGFRvIII AND gate: Left:expression of AND gate is shown recombinant CD19-Fc staining (x-axis)for the CD19 CAR, versus anti-human-Fc staining (Y-axis) for theEGFRvIII CAR. Right: function in response to single positive and doublepositive targets. FIG. 20D. CD19 AND CD5 AND gate: Left: expression ofAND gate is shown recombinant CD19-Fc staining (x-axis) for the CD19CAR, versus anti-human-Fc staining (Y-axis) for the CD5 CAR. Right:function in response to single positive and double positive targets.

FIG. 21A-21C: Function of the AND NOT Gates

Function of the three implementations of an AND NOT gate is shown. Acartoon of the gates tested is shown to the right, and function inresponse to single positive and double positive targets is shown to theleft. FIG. 21A. PTPN6 based AND NOT gate whereby the first CARrecognizes CD19, has a human CD8 stalk spacer and an ITAM containingactivating endodomain; is co-expressed with a second CAR that recognizesCD33, has a mouse CD8 stalk spacer and has an endodomain comprising of aPTPN6 phosphatase domain. FIG. 21B. ITIM based AND NOT gate is identicalto the PTPN6 gate, except the endodomain is replaced by the endodomainfrom LAIR1. FIG. 21C. CD148 boosted AND NOT gate is identical to theITIM based gate except an additional fusion between the PTPN6 SH2 andthe endodomain of CD148 is expressed. All three gates work as expectedwith activation in response to CD19 but not in response to CD19 and CD33together.

FIG. 22A-22C: Dissection of PTPN6 based AND NOT gate function

The original PTPN6 based AND NOT gate is compared with several controlsto demonstrate the model. A cartoon of the gates tested is shown to theright, and function in response to single positive and double positivetargets is shown to the left. FIG. 22A. Original AND NOT gate wherebythe first CAR recognizes CD19, has a human CD8 stalk spacer and an ITAMcontaining activating endodomain; is co-expressed with a second CARrecognizes CD33, has a mouse CD8 stalk spacer and has an endodomaincomprising of a PTPN6 phosphatase domain. FIG. 22B. AND NOT gatemodified so the mouse CD8 stalk spacer is replaced with an Fc spacer.FIG. 22C. AND NOT gate modified so that the PTPN6 phosphatase domain isreplaced with the endodomain from CD148. Original AND NOT gate (FIG.22A) functions as expected triggering in response to CD19, but not inresponse to both CD19 and CD33. The gate in FIG. 22B. triggers both inresponse to CD19 along or CD19 and CD33 together. The gate in FIG. 22C.does not trigger in response to one or both targets.

FIG. 23A-23B: Dissection of LAIR1 based AND NOT gate

Functional activity against CD19 positive, CD33 positive and CD19, CD33double-positive targets is shown. FIG. 23A. Structure and activity ofthe original ITIM based AND NOT gate. This gate is composed of two CARs:the first recognizes CD19, has a human CD8 stalk spacer and an ITAMcontaining endodomain; the second CAR recognizes CD33, has a mouse CD8stalk spacer and an ITIM containing endodomain. FIG. 23B. Structure andactivity of the control ITIM based gate where the mouse CD8 stalk spacerhas been replaced by an Fc domain. This gate is composed of two CARs:the first recognizes CD19, has a human CD8 stalk spacer and an ITAMcontaining endodomain; the second CAR recognizes CD33, has an Fc spacerand an ITIM containing endodomain. Both gates respond to CD19 singlepositive targets, while only the original gate is inactive in responseto CD19 and CD33 double positive targets.

FIG. 24A-24D: Kinetic segregation model of CAR logic gates

Model of kinetic segregation and behaviour of AND gate, NOT AND gate andcontrols. CARs recognize either CD19 or CD33. The immunological synapsecan be imagined between the blue line, which represents the target cellmembrane and the red line, which represents the T-cell membrane. ‘45’ isthe native CD45 protein present on T-cells. ‘H8’ is a CAR ectodomainwith human CD8 stalk as the spacer. ‘Fc’ is a CAR ectodomain with humanHCH2CH3 as the spacer. ‘M8’ is a CAR ectodomain with murine CD8 stalk asthe spacer. ‘19’ represents CD19 on the target cell surface. ‘33’represents CD33 on the target cell surface. The symbol ‘⊕’ represents anactivating endodomain containing ITAMS. The symbol ‘⊖’ represents aphosphatase with slow kinetics—a ‘ligation on’ endodomain such as onecomprising of the catalytic domain of PTPN6 or an ITIM. The symbol ‘0’represents a phosphatase with fast kinetics—a ‘ligation off’ endodomainsuch as the endodomain of CD45 or CD148. This symbol is enlarged in thefigure to emphasize its potent activity.

FIG. 24A. Shows the postulated behaviour of the functional AND gatewhich comprises of a pair of CARs whereby the first CAR recognizes CD19,has a human CD8 stalk spacer and an activating endodomain; and thesecond CAR recognizes CD33, has an Fc spacer and a CD148 endodomain;

FIG. 24B. Shows the postulated behaviour of the control AND gate. Here,the first CAR recognizes CD19, has a human CD8 stalk spacer and anactivating endodomain; and the second CAR recognizes CD33, but has amouse CD8 stalk spacer and a CD148 endodomain;

FIG. 24C. Shows the behaviour of a functional AND NOT gate whichcomprises of a pair of CARs whereby the first CAR recognizes CD19, has ahuman CD8 stalk spacer and an activating endodomain; and the second CARrecognizes CD33, has a mouse CD8 stalk spacer and a PTPN6 endodomain;

FIG. 24D. Shows the postulated behaviour of the control AND NOT gatewhich comprises of a pair of CARs whereby the first CAR recognizes CD19,has a human CD8 stalk spacer and an activating endodomain; and thesecond CAR recognizes CD33, but has an Fc spacer and a PTPN6 endodomain;In the first column, target cells are both CD19 and CD33 negative. Inthe second column, targets are CD19 negative and CD33 positive. In thethird column, target cells are CD19 positive and CD33 negative. In thefourth column, target cells are positive for both CD19 and CD33.

FIG. 25A-25C: Design of APRIL-based CARs.

The CAR design was modified so that the scFv was replaced with amodified form of A proliferation-inducing ligand (APRIL), whichinteracts with interacts with BCMA, TACl and proteoglycans, to act as anantigen binding domain: APRIL was truncated so that the proteoglycanbinding amino-terminus is absent. FIG. 25A. signal peptide was thenattached to truncated APRIL amino-terminus to direct the protein to thecell surface. Three CARs were generated with this APRIL based bindingdomain: FIG. 25A. In the first CAR, the human CD8 stalk domain was usedas a spacer domain. B. In the second CAR, the hinge from IgG1 was usedas a spacer domain. FIG. 25C. In the third CAR, the hinge, CH2 and CH3domains of human IgG1 modified with the pva/a mutations described byHombach et al (2010 Gene Ther. 17:1206-1213) to reduce Fc Receptorbinding was used as a spacer (henceforth referred as Fc-pvaa). In allCARs, these spacers were connected to the CD28 transmembrane domain andthen to a tripartite endodomain containing a fusion of the CD28, OX40and the CD3-Zeta endodomain (Pule et al, Molecular therapy, 2005: Volume12; Issue 5; Pages 933-41).

FIG. 26A-26C: Annotated Amino acid sequence of the above threeAPRIL-CARS

FIG. 26A. Shows the annotated amino acid sequence of the CD8 stalk APRILCAR; FIG. 26B. Shows the annotated amino acid sequence of the APRIL IgG1hinge based CAR; FIG. 26C. Shows the annotated amino acid sequence ofthe APRIL Fc-pvaa based CAR.

FIG. 27A-27C: Expression and ligand binding of different APRIL basedCARs

FIG. 27A. The receptors were co-expressed with a marker gene truncatedCD34 in a retroviral gene vector. Expression of the marker gene ontransduced cells allows confirmation of transduction. FIG. 27B. T-cellswere transduced with APRIL based CARs with either the CD8 stalk spacer,IgG1 hinge or Fc spacer. To test whether these receptors could be stablyexpressed on the cell surface, T-cells were then stained withanti-APRIL-biotin/Streptavidin APC and anti-CD34. Flow-cytometricanalysis was performed. APRIL was equally detected on the cell surfacein the three CARs suggesting they are equally stably expressed. FIG.27C. Next, the capacity of the CARs to recognize TACl and BCMA wasdetermined. The transduced T-cells were stained with either recombinantBCMA or TACl fused to mouse IgG2a Fc fusion along with an anti-mousesecondary and anti-CD34. All three receptor formats showed binding toboth BCMA and TACl. A surprising finding was that binding to BCMA seemedgreater than to TACl. A further surprising finding was that although allthree CARs were equally expressed, the CD8 stalk and IgG1 hinge CARsappeared better at recognizing BCMA and TACl than that with the Fcspacer.

FIG. 28A-28C: Function of the different CAR constructs.

Functional assays were performed with the three different APRIL basedCARs. Normal donor peripheral blood T-cells either non-transduced (NT),or transduced to express the different CARs. Transduction was performedusing equal titer supernatant. These T-cells were then CD56 depleted toremove non-specific NK activity and used as effectors. SupT1 cellseither non-transduced (NT), or transduced to express BCMA or TACl wereused as targets. Data shown is mean and standard deviation from 5independent experiments. FIG. 28A. Specific killing of BCMA and TAClexpressing T-cells was determined using Chromium release. FIG. 28B.Interferon-μ release was also determined. Targets and effectors wereco-cultured at a ratio of 1:1. After 24 hours, Interferon-μ in thesupernatant was assayed by ELISA. FIG. 28C. Proliferation/survival ofCAR T-cells were also determined by counting number of CAR T-cells inthe same co-culture incubated for a further 6 days. All 3 CARs directresponses against BCMA and TACl expressing targets. The responses toBCMA were greater than for TACl.

FIG. 29: AND gate functionality in primary cells.

PBMCs were isolated from blood and stimulated using PHA and IL-2. Twodays later the cells were transduced on retronectin coated plates withretro virus containing the CD19:CD33 AND gate construct. On day 5 theexpression level of the two CARs translated by the AND gate constructwas evaluated via flow cytometry and the cells were depleted of CD56+cells (predominantly NK cells). On day 6 the PBMCs were placed in aco-culture with target cells at a 1:2 effector to target cell ratio. Onday 8 the supernatant was collected and analysed for IFN-gamma secretionvia ELISA

FIG. 30A-30B: FIG. 30A. DNA alignment of the CD28 transmembrane andcytosolic domain of TCRz in the OR gate platform FIG. 30B. Proteinalignment of the CD28 transmembrane and cytosolic domain of TCRz in theOR gate platform.

FIG. 31: Design rules for building logic gated CAR T-cells. OR, AND NOTand AND gated CARs are shown in cartoon format with the target cell ontop, and the T-cell at the bottom with the synapse in the middle. Targetcells express arbitrary target antigens A, and B.

T-cells express two CARs which comprise of anti-A and anti-B recognitiondomains, spacers and endodomains. An OR gate requires (1) spacers simplywhich allow antigen recognition and CAR activation, and (2) both CARs tohave activatory endodomains; An AND NOT gate requires (1) spacers whichresult in co-segregation of both CARs upon recognition of both antigensand (2) one CAR with an activatory endodomain, and the other whoseendodomain comprises or recruits a weak phosphatase; An AND gaterequires (1) spacers which result in segregation of both CARs intodifferent parts of the immunological synapse upon recognition of bothantigens and (2) one CAR with an activatory endodomain, and the otherwhose endodomain comprises of a potent phosphatase.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a panel of “logic-gated” chimericantigen receptor pairs which, when expressed by a cell, such as a Tcell, are capable of detecting a particular pattern of expression of atleast two target antigens. If the at least two target antigens arearbitrarily denoted as antigen A and antigen B, the three possibleoptions are as follows:

“OR GATE”—T cell triggers when either antigen A or antigen B is presenton the target cell “AND GATE”—T cell triggers only when both antigens Aand B are present on the target cell “AND NOT GATE”—T cell triggers ifantigen A is present alone on the target cell, but not if both antigensA and B are present on the target cell

Engineered T cells expressing these CAR combinations can be tailored tobe exquisitely specific for cancer cells, based on their particularexpression (or lack of expression) of two or more markers.

Thus in a first aspect, the present invention provides a cell whichco-expresses a first chimeric antigen receptor (CAR) and second CAR atthe cell surface, each CAR comprising:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer    -   (iii) a trans-membrane domain; and    -   (iv) an intracellular T cell signaling domain (endodomain)

wherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first or second CARs is anactivating CAR comprising an activating intracellular T cell signalingdomain.

The cell may be an immune effector cell, such as a T-cell or naturalkiller (NK) cell. Features mentioned herein in connection with a T cellapply equally to other immune effector cells, such as NK cells.

The spacer of the first CAR may be different to the spacer of the secondCAR. such that the first and second CARs do not form heterodimers

The spacer of the first CAR may have a different length and/or sizeand/or configuration from the spacer of the second CAR, such that eachCAR is tailored for recognition of its respective target antigen.

The spacer of the first CAR may have a different length and/or chargeand/or shape and/or configuration and/or glycosylation to the spacer ofthe second CAR, such that when the first CAR and the second CAR bindtheir respective target antigens, the first CAR and second CAR becomespatially separated on the T cell. Ligation of the first and second CARsto their respective antigens causes them to be compartmentalizedtogether or separately in the immunological synapse resulting in controlof activation. This may be understood when one considers the kineticseparation model of T-cell activation (see below).

The first spacer or the second spacer may comprise a CD8 stalk and theother spacer may comprise the hinge, CH2 and CH3 domain of an IgG1.

In the present invention, relating to the “OR” gate, both the first andsecond CAR are activating CARs. An activating CAR comprises anactivating endodomain, such that it causes T cell activation uponantigen binding. Since the OR gate comprises first and second CARs whichare both activating, T cell activation occurs when a target cellexpresses either or both target antigens.

Either the first or the second CAR may bind CD19 and the other CAR maybind CD20. This is advantageous because some lymphomas and leukemiasbecome CD19 negative after CD19 targeting, so it gives a “back-up”antigen, should this occur.

In a second aspect, the present invention provides a nucleic acidsequence encoding both the first and second chimeric antigen receptors(CARs) as defined in the first aspect of the invention.

The nucleic acid sequence according may have the following structure:AgB1-spacer1-TM1-endo1-coexpr-AgB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR; spacer 1 is a nucleic acid sequence encoding the spacerof the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR; endo 1 is a nucleic acid sequence encoding the endodomain ofthe first CAR;

coexpr is a nucleic acid sequence allowing co-expression of two CARs(e.g. a cleavage site);

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a a nucleic acid sequence encoding the transmembrane domain ofthe second CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR;

which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

The nucleic acid sequence allowing co-expression of two CARs may encodea self-cleaving peptide or a sequence which allows alternative means ofco-expressing two CARs such as an internal ribosome entry sequence or a2^(nd) promoter or other such means whereby one skilled in the art canexpress two proteins from the same vector.

Alternative codons may be used in regions of sequence encoding the sameor similar amino acid sequences, such as the transmembrane and/orintracellular T cell signalling domain (endodomain) in order to avoidhomologous recombination. An example of such “codon wobbling” is shownin FIG. 30.

In a third aspect, the present invention provides a kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR) as defined in the first aspect of the        invention, which nucleic acid sequence has the following        structure:

AgB1-spacer1-TM1-endo1

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR; and

-   -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor

(CAR) as defined in the first aspect of the invention, which nucleicacid sequence has the following structure:

Ag B2-spacer2-TM2-endo2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR.

In a fourth aspect, the present invention provides a kit comprising: afirst vector which comprises the first nucleic acid sequence as definedabove; and a second vector which comprises the first nucleic acidsequence as defined above.

The vectors may be plasmid vectors, retroviral vectors or transposonvectors. The vectors may be lentiviral vectors.

In a fifth aspect, the present invention provides a vector comprising anucleic acid sequence according to the second aspect of the invention.The vector may be a lentiviral vector.

The vector may be a plasmid vector, a retroviral vector or a transposonvector.

In a sixth aspect, the present invention involves co-expressing morethan two CARs in such a fashion that a complex pattern of more than twoantigens can be recognized on the target cell.

In a seventh aspect, the present invention provides a method for makinga T cell according to the first aspect of the invention, which comprisesthe step of introducing one or more nucleic acid sequence (s) encodingthe first and second CARs; or one or more vector(s) as defined aboveinto a T cell.

The T cell may be from a sample isolated from a patient, a related orunrelated haematopoietic transplant donor, a completely unconnecteddonor, from cord blood, differentiated from an embryonic cell line,differentiated from an inducible progenitor cell line, or derived from atransformed T cell line.

In an eighth aspect, the present invention provides a pharmaceuticalcomposition comprising a plurality of T cells according to the firstaspect of the invention.

In a ninth aspect, the present invention provides a method for treatingand/or preventing a disease, which comprises the step of administering apharmaceutical composition according to the eighth aspect of theinvention to a subject.

The method may comprise the following steps:

-   -   (i) isolation of a T cell as listed above.    -   (ii) transduction or transfection of the T cells with one or        more nucleic acid sequence(s) encoding the first and second CAR        or one or more vector(s) comprising such nucleic acid        sequence(s); and    -   (iii) administering the T cells from (ii) to the subject.

The disease may be a cancer.

In a tenth aspect, the present invention provides a pharmaceuticalcomposition according to the eighth aspect of the invention for use intreating and/or preventing a disease.

The disease may be a cancer.

In an eleventh aspect, the present invention provides use of a T cellaccording to the first aspect of the invention in the manufacture of amedicament for treating and/or preventing a disease.

The disease may be a cancer.

The present invention also provides a nucleic acid sequence whichcomprises:

a) a first nucleotide sequence encoding a first chimeric antigenreceptor (CAR);

b) a second nucleotide sequence encoding a second CAR;

c) a sequence encoding a self-cleaving peptide positioned between thefirst and second nucleotide sequences, such that the two CARs areexpressed as separate entities.

Alternative codons may be used in one or more portion(s) of the firstand second nucleotide sequences in regions which encode the same orsimilar amino acid sequence(s).

The present invention also provides a vector and a cell comprising sucha nucleic acid.

By providing two or more CARs on the surface of the T cell, it ispossible to target multiple cancer markers simultaneously, providingbetter therapeutic efficacy for heterogeneic tumours and avoiding theproblem of cancer escape.

Because the CARs are expressed on the surface of the T cell as separatemolecules, this approach overcomes the spatial and accessibility issuesassociated with TanCARs. T-cell activation efficiency is also improved.As each CAR has its own spacer, it is possible to tailor the spacer andtherefore the distance that the binding domain projects from the T cellsurface and its flexibility etc to the particular target antigen. Thischoice is unfettered by the design considerations associated withTanCARs, i.e. that one CAR needs to be juxta-posed to the T cellmembrane and one CAR needs to be distal, positioned in tandem to thefirst CAR.

By providing a single nucleic acid which encodes the two CARs separatedby a cleavage site, it is possible to engineer T cells to co-express thetwo CARs using a simple single transduction procedure. A doubletransfection procedure could be used with CAR-encoding sequences inseparate constructs, but this would be more complex and expensive andrequires more integration sites for the nucleic acids. A doubletransfection procedure would also be associated with uncertainty as towhether both CAR-encoding nucleic acids had been transduced andexpressed effectively. This is especially true for a multiple CARapproach where three or more CARs are introduced to the cell.

The two or more CARs will have portions of high homology, for examplethe transmembrane and/or intracellular signalling domains are likely tobe highly homologous. If the same or similar linkers are use for the twoCARs, then they will also be highly homologous. This would suggest thatan approach where both CARs are provided on a single nucleic acidsequence would be inappropriate, because of the likelihood of homologousrecombination between the sequences. However, the present inventors havefound that by “codon wobbling” the portions of sequence encoding areasof high homology, it is possible to express two CARs from a singleconstruct with high efficiency. Codon wobbling involves usingalternative codons in regions of sequence encoding the same or similaramino acid sequences.

FURTHER ASPECTS OF THE INVENTION

The present invention also relates to the aspects listed in thefollowing numbered paragraphs:

1. A T cell which co-expresses a first chimeric antigen receptor (CAR)and second CAR at the cell surface, each CAR comprising:

-   -   (i) an antigen-binding domain;    -   (ii) a spacer    -   (iii) a trans-membrane domain; and    -   (iv) an endodomain

wherein the antigen binding domains of the first and second CARs bind todifferent antigens, wherein the spacer of the first CAR is different tothe spacer of the second CAR and wherein one of the first or second CARsis an activating CAR comprising an activating endodomain and the otherCAR is either an activating CAR comprising an activating endodomain oran inhibitory CAR comprising a ligation-on or ligation-off inhibitoryendodomain.

2. A T cell according to paragraph 1, wherein the spacer of the firstCAR has a different length and/or charge and/or size and/orconfiguration and/or glycosylation of the spacer of the second CAR, suchthat when the first CAR and the second CAR bind their respective targetantigens, the first CAR and second CAR become spatially separated on theT cell membrane.

3. A T cell according to paragraph 2, wherein either the first spacer orthe second spacer comprises a CD8 stalk and the other spacer comprisesthe hinge, CH2 and CH3 domain of IgG1.

4. A T cell according to paragraph 1, wherein both the first and secondCARs are activating CARs.

5. A T cell according to paragraph 4, wherein one CAR binds CD19 and theother CAR binds CD20.

6. A T cell according to paragraph 2 or 3, wherein one of the first orsecond CARs is an activating CAR comprising an activating endodomain,and the other CAR is an inhibitory CAR comprising a ligation-offinhibitory endodomain, which inhibitory CAR inhibits T-cell activationby the activating CAR in the absence of inhibitory CAR ligation, butdoes not significantly inhibit T-cell activation by the activating CARwhen the inhibitory CAR is ligated.

7. A T cell according to paragraph 6, wherein the inhibitory endodomaincomprises all or part of the endodomain from CD148 or CD45.

8. A T cell according to paragraph 6 or 7, wherein the antigen-bindingdomain of the first CAR binds CD5 and the antigen-binding domain of thesecond CAR binds CD19.

9. A T cell according to paragraph 1 wherein the first and secondspacers are sufficiently different so as to prevent cross-pairing of thefirst and second CARs but are sufficiently similar to result inco-localisation of the first and second CARs following ligation.

10. A T cell according to paragraph 9, wherein one of the first orsecond CARs in an activating CAR comprising an activating endodomain,and the other CAR is an inhibitory CAR comprising a ligation-oninhibitory endodomain, which inhibitory CAR does not significantlyinhibit T-cell activation by the activating CAR in the absence ofinhibitory CAR ligation, but inhibits T-cell activation by theactivating CAR when the inhibitory CAR is ligated.

11. A T cell according to paragraph 10, wherein the ligation-oninhibitory endodomain comprises at least part of a phosphatase.

12. A T cell according to paragraph 11, wherein the ligation-oninhibitory endodomain comprises all or part of PTPN6.

13. A T cell according to paragraph 10, wherein the ligation-oninhibitory endodomain comprises at least one ITIM domain.

14. A T cell according to paragraph 13, wherein activity of theligation-on inhibitory endodomain is enhanced by co-expression of aPTPN6-CD45 or -CD148 fusion protein.

15. A T cell according to any of paragraphs 10 to 14, wherein the CARcomprising the activating endodomain comprises an antigen-binding domainwhich binds CD33 and the CAR which comprises the ligation-on inhibitoryendodomain comprises an antigen-binding domain which binds CD34.

16. A T cell which comprises more than two CARs as defined in thepreceding paragraphs such that it is specifically stimulated by a cell,such as a T cell, bearing a distinct pattern of more than two antigens.

17. A nucleic acid sequence encoding both the first and second chimericantigen receptors (CARs) as defined in any of paragraphs 1 to 16.

18. A nucleic acid sequence according to paragraph 17, which has thefollowing structure:

AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR;

coexpr is a nucleic acid sequence enabling co-expression of both CARs

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR;

spacer 2 is a nucleic acid sequence encoding the spacer of the secondCAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR;

which nucleic acid sequence, when expressed in a T cell, encodes apolypeptide which is cleaved at the cleavage site such that the firstand second CARs are co-expressed at the T cell surface.

19. A nucleic acid sequence according to paragraph 18, wherein coexprencodes a sequence comprising a self-cleaving peptide.

20. A nucleic acid sequence according to paragraph 18 or 19, whereinalternative codons are used in regions of sequence encoding the same orsimilar amino acid sequences, in order to avoid homologousrecombination.

21. A kit which comprises

-   -   (i) a first nucleic acid sequence encoding the first chimeric        antigen receptor (CAR) as defined in any of paragraphs 1 to 16,        which nucleic acid sequence has the following structure:

AgB1-spacer1-TM1-endo1

in which

AgB1 is a nucleic acid sequence encoding the antigen-binding domain ofthe first CAR;

spacer 1 is a nucleic acid sequence encoding the spacer of the firstCAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of thefirst CAR;

endo 1 is a nucleic acid sequence encoding the endodomain of the firstCAR; and

-   -   (ii) a second nucleic acid sequence encoding the second chimeric        antigen receptor (CAR) as defined in any of paragraphs 1 to 16,        which nucleic acid sequence has the following structure:

AgB2-spacer2-TM2-endo2

AgB2 is a nucleic acid sequence encoding the antigen-binding domain ofthe second CAR; spacer 2 is a nucleic acid sequence encoding the spacerof the second CAR;

TM2 is a nucleic acid sequence encoding the transmembrane domain of thesecond CAR;

endo 2 is a nucleic acid sequence encoding the endodomain of the secondCAR.

22. A kit comprising: a first vector which comprises the first nucleicacid sequence as defined in paragraph 21; and a second vector whichcomprises the first nucleic acid sequence as defined in paragraph 21.

23. A kit according to paragraph 22, wherein the vectors are integratingviral vectors or transposons.

24. A vector comprising a nucleic acid sequence according to any ofparagraphs 17 to 20.

25. A retroviral vector or a lentiviral vector or a transposon accordingto paragraph 24.

26. A method for making a T cell according to any of paragraphs 1 to 16,which comprises the step of introducing: a nucleic acid sequenceaccording to any of paragraphs 17 to 20; a first nucleic acid sequenceand a second nucleic acid sequence as defined in paragraph 21; and/or afirst vector and a second vector as defined in paragraph 22 or a vectoraccording to paragraph 24 or 25, into a T cell.

27. A method according to paragraph 24, wherein the T cell is from asample isolated from a subject.

28. A pharmaceutical composition comprising a plurality of T cellsaccording to any of paragraphs 1 to 16.

29. A method for treating and/or preventing a disease, which comprisesthe step of administering a pharmaceutical composition according toparagraph 28 to a subject.

30. A method according to paragraph 29, which comprises the followingsteps:

-   -   (i) isolation of a T cell-containing sample from a subject;    -   (ii) transduction or transfection of the T cells with: a nucleic        acid sequence according to any of paragraphs 17 to 20; a first        nucleic acid sequence and a second nucleic acid sequence as        defined in paragraph 21; a first vector and a second vector as        defined in paragraph 22 or 23 or a vector according to paragraph        24 or 25; and    -   (iii) administering the T cells from (ii) to a the subject.

31. A method according to paragraph 29 or 30, wherein the disease is acancer.

32. A pharmaceutical composition according to paragraph 28 for use intreating and/or preventing a disease.

33. The use of a T cell according to any of paragraphs 1 to 16 in themanufacture of a medicament for treating and/or preventing a disease.

DETAILED DESCRIPTION

Chimeric Antigen Receptors (CARs)

CARs, which are shown schematically in FIG. 1, are chimeric type Itrans-membrane proteins which connect an extracellularantigen-recognizing domain (binder) to an intracellular signallingdomain (endodomain). The binder is typically a single-chain variablefragment (scFv) derived from a monoclonal antibody (mAb), but it can bebased on other formats which comprise an antibody-like antigen bindingsite. A spacer domain is usually necessary to isolate the binder fromthe membrane and to allow it a suitable orientation. A common spacerdomain used is the Fc of IgG1. More compact spacers can suffice e.g. thestalk from CD8a and even just the IgG1 hinge alone, depending on theantigen. A trans-membrane domain anchors the protein in the cellmembrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular partsof either the γ chain of the FcεR1 or CD3ζ. Consequently, these firstgeneration receptors transmitted immunological signal 1, which wassufficient to trigger T-cell killing of cognate target cells but failedto fully activate the T-cell to proliferate and survive. To overcomethis limitation, compound endodomains have been constructed: fusion ofthe intracellular part of a T-cell co-stimulatory molecule to that ofCD3ζ results in second generation receptors which can transmit anactivating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used is that ofCD28. This supplies the most potent co-stimulatory signal—namelyimmunological signal 2, which triggers T-cell proliferation. Somereceptors have also been described which include TNF receptor familyendodomains, such as the closely related OX40 and 41 BB which transmitsurvival signals. Even more potent third generation CARs have now beendescribed which have endodomains capable of transmitting activation,proliferation and survival signals.

CAR-encoding nucleic acids may be transferred to T cells using, forexample, retroviral vectors. Lentiviral vectors may be employed. In thisway, a large number of cancer-specific T cells can be generated foradoptive cell transfer. When the CAR binds the target-antigen, thisresults in the transmission of an activating signal to the T-cell it isexpressed on. Thus the CAR directs the specificity and cytotoxicity ofthe T cell towards tumour cells expressing the targeted antigen.

The first aspect of the invention relates to a T-cell which co-expressesa first CAR and a second CAR such that a T-cell can recognize a desiredpattern of expression on target cells in the manner of a logic gate asdetailed in the truth tables: table 1, 2 and 3.

Both the first and second (and optionally subsequent) CARs comprise:

(i) an antigen-binding domain;

(ii) a spacer;

(iii) a transmembrane domain; and

(iii) an intracellular domain.

TABLE 1 Truth Table for CAR OR GATE Antigen A Antigen B Response AbsentAbsent No activation Absent Present Activation Present Absent ActivationPresent Present Activation

TABLE 2 Truth Table for CAR AND GATE Antigen A Antigen B Response AbsentAbsent No activation Absent Present No Activation Present Absent NoActivation Present Present Activation

TABLE 3 Truth Table for CAR AND NOT GATE Antigen A Antigen B ResponseAbsent Absent No activation Absent Present No Activation Present AbsentActivation Present Present No Activation

The first and second CAR of the T cell of the present invention may beproduced as a polypeptide comprising both CARs, together with a cleavagesite.

SEQ ID No. 1 to 5 give examples of such polypeptides, which eachcomprise two CARs. The CAR may therefore comprise one or other part ofthe following amino acid sequences, which corresponds to a single CAR.

SEQ ID No 1 is a CAR OR gate which recognizes CD19 OR CD33

SEQ ID No 2 Is a CAR AND gate which recognizes CD19 AND CD33 using aCD148 phosphatase

SEQ ID No 3 Is an alternative implementation of the CAR AND GATE whichrecognizes CD19 AND CD33 which uses a CD45 phosphatase

SEQ ID No 4 Is a CAR AND NOT GATE which recognizes CD19 AND NOT CD33based on PTPN6 phosphatase

SEQ ID No 5 Is an alternative implementation of the CAR AND NOT gatewhich recognizes CD19 AND NOT CD33 and is based on an ITIM containingendodomain from LAIR1

SEQ ID No 6. Is a further alternative implementation of the CAR AND NOTgate which recognizes CD19 AND NOT CD33 and recruits a PTPN6-CD148fusion protein to an ITIM containing endodomain.

SEQ ID No. 1 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R SEQ ID No. 2MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA SEQ ID No. 3MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS SEQ ID No. 4MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYWAPLAGICVALLLSLIITLICYHRSRKRVCKSGGGSFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAI AQFIETTKKKLSEQ ID No. 5 MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH SEQ ID No. 6MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMAVPIQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYDTNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSGGGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVIYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARHRAEGRGSLLTCGDVEENPGPWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYSGGGGSFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIASGS

The CAR may comprise a variant of the CAR-encoding part of the sequenceshown as SEQ ID No. 1, 2, 3, 4, 5 or 6 having at least 80, 85, 90, 95,98 or 99% sequence identity, provided that the variant sequence is a CARhaving the required properties.

Methods of sequence alignment are well known in the art and areaccomplished using suitable alignment programs. The % sequence identityrefers to the percentage of amino acid or nucleotide residues that areidentical in the two sequences when they are optimally aligned.Nucleotide and protein sequence homology or identity may be determinedusing standard algorithms such as a BLAST program (Basic Local AlignmentSearch Tool at the National Center for Biotechnology Information) usingdefault parameters, which is publicly available athttp://blast.ncbi.nlm.nih.gov. Other algorithms for determining sequenceidentity or homology include: LALIGN(http://www.ebi.ac.uk/Tools/psa/Ialign/ andhttp://www.ebi.ac.uk/Tools/psa/Ialign/nucleotide.html), AMAS (Analysisof Multiply Aligned Sequences, athttp://www.compbio.dundee.ac.uk/Softwar/Amas/amas.html), FASTA(http://ebi.ac.uk/Tools/sss/fasta/) Clustal Omega(http://www.ebi.ac.uk.Tools/msa/clustalo/), SIM(http://web.expasy.org/sim/), and EMBOSS Needle(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.ht,l).

Car Logical or Gate

In this embodiment, the antigen binding domains of the first and secondCARs of the present invention bind to different antigens and both CARscomprise an activating endodomain. The spacer domains may be the same,or sufficiently different to prevent cross-pairing of the two differentreceptors. A T cell can hence be engineered to activate upon recognitionof either or both antigens. This is useful in the field of oncology asindicated by the Goldie-Coldman hypothesis: sole targeting of a singleantigen may result in tumour escape by modulation of said antigen due tothe high mutation rate inherent in most cancers. By simultaneouslytargeting two antigens, the probably of such escape is exponentiallyreduced.

Various tumour associated antigens are known as shown in the followingTable 4. For a given disease, the first CAR and second CAR may bind totwo different TAAs associated with that disease. In this way, tumourescape by modulating a single antigen is prevented, since a secondantigen is also targeted. For example, when targeting a B-cellmalignancy, both CD19 and CD20 can be simultaneously targeted. In thisembodiment, it is important that the two CARs do not heterodimerize.

TABLE 4 Cancer type TAA Diffuse Large B-cell Lymphoma CD19, CD20 Breastcancer ErbB2, MUC1 AML CD13, CD33 Neuroblastoma GD2, NCAM B-CLL CD19,CD52 Colorectal cancer Folate binding protein, CA-125

Kinetic Segregation Model

Subsequent pairing of CARs to generate the AND gate and the AND NOT gateare based on the kinetic segregation model (KS) of T-cell activation.This is a functional model, backed by experimental data, which explainshow antigen recognition by a T-cell receptor is converted intodown-stream activation signals. Briefly: at the ground state, thesignalling components on the T-cell membrane are in dynamic homeostasiswhereby dephosphorylated ITAMs are favoured over phosphorylated ITAMs.This is due to greater activity of the transmembrane CD45/CD148phosphatases over membrane-tethered kinases such as Ick. When a T-cellengages a target cell through a T-cell receptor (or CAR) recognition ofcognate antigen, tight immunological synapses form. This closejuxtapositioning of the T-cell and target membranes excludes CD45/CD148due to their large ectodomains which cannot fit into the synapse.Segregation of a high concentration of T-cell receptor associated ITAMsand kinases in the synapse, in the absence of phosphatases, leads to astate whereby phosphorylated ITAMs are favoured. ZAP70 recognizes athreshold of phosphorylated ITAMs and propagates a T-cell activationsignal. This advanced understanding of T-cell activation is exploited bythe present invention. In particular, the invention is based on thisunderstanding of how ectodomains of different length and/or bulk and/orcharge and/or configuration and/or glycosylation result in differentialsegregation upon synapse formation.

The Car Logical and Gate

In this embodiment, one CAR comprises an activating endodomain and oneCAR comprises an inhibitory endodomain whereby the inhibitory CARconstitutively inhibits the first activating CAR, but upon recognitionof its cognate antigen releases its inhibition of the activating CAR. Inthis manner, a T-cell can be engineered to trigger only if a target cellexpresses both cognate antigens. This behaviour is achieved by theactivating CAR comprising an activating endodomain containing ITAMdomains for example the endodomain of CD3 Zeta, and the inhibitory CARcomprising the endodomain from a phosphatase able to dephosphorylate anITAM (e.g. CD45 or CD148). Crucially, the spacer domains of both CARsare significantly different in size and/or shape and/or charge etc. Whenonly the activating CAR is ligated, the inhibitory CAR is in solution onthe T-cell surface and can diffuse in and out of the synapse inhibitingthe activating CAR. When both CARs are ligated, due to differences inspacer properties, the activating and inhibiting CAR are differentiallysegregated allowing the activating CAR to trigger T-cell activationunhindered by the inhibiting CAR.

This is of considerable utility in the field of cancer therapy.Currently, immunotherapies typically target a single antigen. Mostcancers cannot be differentiated from normal tissues on the basis of asingle antigen. Hence, considerable “on-target off-tumour” toxicityoccurs whereby normal tissues are damaged by the therapy. For instance,whilst targeting CD20 to treat B-cell lymphomas with Rituximab, theentire normal B-cell compartment is depleted. For instance, whilsttargeting CD52 to treat chronic lymphocytic leukaemia, the entirelymphoid compartment is depleted. For instance, whilst targeting CD33 totreat acute myeloid leukaemia, the entire myeloid compartment is damagedetc. By restricting activity to a pair of antigens, much more refinedtargeting, and hence less toxic therapy can be developed. A practicalexample is targeting of CLL which expresses both CD5 and CD19. Only asmall proportion of normal B-cells express both antigens, so theoff-target toxicity of targeting both antigens with a logical AND gateis substantially less than targeting each antigen individually.

The design of the present invention is a considerable improvement onprevious implementation as described by Wilkie et al. ((2012). J. Clin.Immunol. 32, 1059-1070) and then tested in vivo (Kloss et al (2013) Nat.Biotechnol. 31, 71-75). In this implementation, the first CAR comprisesof an activating endodomain, and the second a co-stimulatory domain.This way, a T-cell only receives an activating and co-stimulatory signalwhen both antigens are present. However, the T-cell still will activatein the sole presence of the first antigen resulting in the potential foroff-target toxicity. Further, the implementation of the presentinvention allows for multiple compound linked gates whereby a cell caninterpret a complex pattern of antigens.

TABLE 5 Cancer Type Antigens Chronic Lymphocytic Leukaemia CD5, CD19Neuroblastoma ALK, GD2 Glioma EGFR, Vimentin Multiple myeloma BCMA,CD138 Renal Cell Carcinoma Carbonic anhydrase IX, G250 T-ALL CD2,N-Cadherin Prostate Cancer PSMA, hepsin (or others)

The Car Logical and not Gate

In this embodiment, one CAR comprises an activating endodomain and oneCAR comprises an inhibitory endodomain such that this inhibitory CAR isonly active when it recognizes its cognate antigen. Hence a T-cellengineered in this manner is activated in response to the sole presenceof the first antigen but is not activated when both antigens arepresent. This invention is implemented by inhibitory CARs with a spacerthat co-localise with the first CAR but either the phosphatase activityof the inhibitory CAR should not be so potent that it inhibits insolution, or the inhibitory endodomain in fact recruits a phosphatasesolely when the inhibitory CAR recognizes its cognate target. Suchendodomains are termed “ligation-on” or semi-inhibitory herein.

This invention is of use in refining targeting when a tumour can bedistinguished from normal tissue by the presence of tumour associatedantigen and the loss of an antigen expressed on normal tissue. The ANDNOT gate is of considerable utility in the field of oncology as itallows targeting of an antigen which is expressed by a normal cell,which normal cell also expresses the antigen recognised by the CARcomprising the activating endodomain. An example of such an antigen isCD33 which is expressed by normal stem cells and acute myeloid leukaemia(AML) cells. CD34 is expressed on stem cells but not typically expressedon AML cells. A T-cell recognizing CD33 AND NOT CD34 would result indestruction of leukaemia cells but sparing of normal stem cells.

Potential antigen pairs for use with AND NOT gates are shown in Table 6.

TABLE 6 Antigen expressed Normal cell which by normal cell but DiseaseTAA expresses TAA not cancer cell AML CD33 stem cells CD34 Myeloma BCMADendritic cells CD1c B-CLL CD160 Natural Killer cells CD56 Prostatecancer PSMA Neural Tissue NCAM Bowel cancer A33 Normal bowel HLA class Iepithelium

Compound Gates

The kinetic segregation model with the above components allows compoundgates to be made e.g. a T-cell which triggers in response to patterns ofmore than two target antigens. For example, it is possible to make a Tcell which only triggers when three antigens are present (A AND B ANDC). Here, a cell expresses three CARs, each recognizing antigens A, Band C. One CAR is excitatory and two are inhibitory, which each CARhaving spacer domains which result in differential segregation. Onlywhen all three are ligated, will the T-cell activate. A further example:(A OR B) AND C: here, CARs recognizing antigens A and B are activatingand have spacers which co-localise, while CAR recognizing antigen C isinhibitory and has a spacer which results in different co-segregation. Afurther example (A AND NOT B) AND C: Here CAR against antigen A has anactivating endodomain and co-localises with CAR against antigen B whichhas a conditionally inhibiting endodomain. CAR against antigen C has aspacer who segregates differently from A or B and is inhibitory. Infact, ever more complex boolean logic can be programmed with thesesimple components of the invention with any number of CARs and spacers.

Signal Peptide

The CARs of the T cell of the present invention may comprise a signalpeptide so that when the CAR is expressed inside a cell, such as aT-cell, the nascent protein is directed to the endoplasmic reticulum andsubsequently to the cell surface, where it is expressed.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The signal peptide may comprise the SEQ ID No. 7, 8 or 9 or a variantthereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions,substitutions or additions) provided that the signal peptide stillfunctions to cause cell surface expression of the CAR.

SEQ ID No. 7: MGTSLLCWMALCLLGADHADG 

The signal peptide of SEQ ID No. 7 is compact and highly efficient. Itis predicted to give about 95% cleavage after the terminal glycine,giving efficient removal by signal peptidase.

SEQ ID No. 8: MSLPVTALLLPLALLLHAARP 

The signal peptide of SEQ ID No. 8 is derived from IgG1.

SEQ ID No. 9: MAVPTQVLGLLLLWLTDARC 

The signal peptide of SEQ ID No. 9 is derived from CD8.

The signal peptide for the first CAR may have a different sequence fromthe signal peptide of the second CAR (and from the 3^(rd) CAR and 4^(th)CAR etc).

Antigen Binding Domain

The antigen binding domain is the portion of the CAR which recognizesantigen. Numerous antigen-binding domains are known in the art,including those based on the antigen binding site of an antibody,antibody mimetics, and T-cell receptors. For example, theantigen-binding domain may comprise: a single-chain variable fragment(scFv) derived from a monoclonal antibody; a natural ligand of thetarget antigen; a peptide with sufficient affinity for the target; asingle domain antibody; an artificial single binder such as a Darpin(designed ankyrin repeat protein); or a single-chain derived from aT-cell receptor.

The antigen binding domain may comprise a domain which is not based onthe antigen binding site of an antibody. For example the antigen bindingdomain may comprise a domain based on a protein/peptide which is asoluble ligand for a tumour cell surface receptor (e.g. a solublepeptide such as a cytokine or a chemokine); or an extracellular domainof a membrane anchored ligand or a receptor for which the binding paircounterpart is expressed on the tumour cell.

Examples 11 to 13 relate to a CAR which binds BCMA, in which the antigenbinding doaimn comprises APRIL, a ligand for BCMA.

The antigen binding domain may be based on a natural ligand of theantigen.

The antigen binding domain may comprise an affinity peptide from acombinatorial library or a de novo designed affinity protein/peptide.

Spacer Domain

CARs comprise a spacer sequence to connect the antigen-binding domainwith the transmembrane domain and spatially separate the antigen-bindingdomain from the endodomain. A flexible spacer allows the antigen-bindingdomain to orient in different directions to facilitate binding.

In the T cell of the present invention, the first and second CARscomprise different spacer molecules. For example, the spacer sequencemay, for example, comprise an IgG1 Fc region, an IgG1 hinge or a humanCD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprisean alternative linker sequence which has similar length and/or domainspacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. Ahuman IgG1 spacer may be altered to remove Fc binding motifs.

Examples of amino acid sequences for these spacers are given below:

(hinge-CH2CH3 of human IgG1) SEQ ID No. 10AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD (human CD8 stalk): SEQ ID No. 11ITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHIRGLDFACDI (human IgG1 hinge):SEQ ID No. 12 AEPKSPDKTHTCPPCPKDPK (CD2 ectodomain) SEQ ID No. 13KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQERVSKPKISWTCINTTLTCEVMNGTDPELNLYQDGKHLKLSQRVITHKWTTSLSAKFKCTAGNKVSKESSVEPVSCPEKGLD (CD34 ectodomain) SEQ ID No. 14SLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTSTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLEQNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVA SHQSYSQKT

Since CARs are typically homodimers (see FIG. 1a ), cross-pairing mayresult in a heterodimeric chimeric antigen receptor. This is undesirablefor various reasons, for example: (1) the epitope may not be at the same“level” on the target cell so that a cross-paired CAR may only be ableto bind to one antigen; (2) the VH and VL from the two different scFvcould swap over and either fail to recognize target or worse recognizean unexpected and unpredicted antigen. For the “OR” gate and the “ANDNOT” gate, the spacer of the first CAR may be sufficiently differentfrom the spacer of the second CAR in order to avoid cross-pairing. Theamino acid sequence of the first spacer may share less that 50%, 40%,30% or 20% identity at the amino acid level with the second spacer.

In other aspects of the invention (for example the AND gate) it isimportant that the spacer of the first CAR has a different length,and/or charge and/or shape and/or configuration and/or glycosylation,such that when both first and second CARs bind their target antigen, thedifference in spacer charge or dimensions results in spatial separationof the two types of CAR to different parts of the membrane to result inactivation as predicted by the kinetic separation model. In theseaspects, the different length, shape and/or configuration of the spacersis carefully chosen bearing in mind the size and binding epitope on thetarget antigen to allow differential segregation upon cognate targetrecognition. For example the IgG1 Hinge, CD8 stalk, IgG1 Fc, ectodomainof CD34, ectodomain of CD45 are expected to differentially segregate.

Examples of spacer pairs which differentially segregate and aretherefore suitable for use with the AND gate are shown in the followingTable:

Stimulatory CAR spacer Inhibitory CAR spacer Human-CD8STKHuman-IgG-Hinge-CH2CH3 Human-CD3z ectodomain Human-IgG-Hinge-CH2CH3Human-IgG-Hinge Human-IgG-Hinge-CH2CH3 Human-CD28STKHuman-IgG-Hinge-CH2CH3 Human-CD8STK Human-IgM-Hinge-CH2CH3CD4 Human-CD3zectodomain Human-IgM-Hinge-CH2CH3CD4 Human-IgG-HingeHuman-IgM-Hinge-CH2CH3CD4 Human-CD28STK Human-IgM-Hinge-CH2CH3CD4

In other aspects of the invention (for example the AND NOT gate), it isimportant that the spacer be sufficiently different as to preventcross-pairing, but to be sufficiently similar to co-localise. Pairs oforthologous spacer sequences may be employed. Examples are murine andhuman CD8 stalks, or alternatively spacer domains which aremonomeric—for instance the ectodomain of CD2.

Examples of spacer pairs which co-localise and are therefore suitablefor use with the AND NOT gate are shown in the following Table:

Stimulatory CAR spacer Inhibitory CAR spacer Human-CD8aSTK Mouse CD8aSTKHuman-CD28STK Mouse CD8aSTK Human-IgG-Hinge Human-CD3z ectodomainHuman-CD8aSTK Mouse CD28STK Human-CD28STK Mouse CD28STKHuman-IgG-Hinge-CH2CH3 Human-IgM-Hinge-CH2CH3CD4

All the spacer domains mentioned above form homodimers. However themechanism is not limited to using homodimeric receptors and should workwith monomeric receptors as long as the spacer is sufficiently rigid. Anexample of such a spacer is CD2 or truncated CD22.

Transmembrane Domain

The transmembrane domain is the sequence of the CAR that spans themembrane.

A transmembrane domain may be any protein structure which isthermodynamically stable in a membrane. This is typically an alpha helixcomprising of several hydrophobic residues. The transmembrane domain ofany transmembrane protein can be used to supply the transmembraneportion of the invention. The presence and span of a transmembranedomain of a protein can be determined by those skilled in the art usingthe TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/).Further, given that the transmembrane domain of a protein is arelatively simple structure, i.e. a polypeptide sequence predicted toform a hydrophobic alpha helix of sufficient length to span themembrane, an artificially designed TM domain may also be used (U.S. Pat.No. 7,052,906 B1 describes synthetic transmembrane components).

The transmembrane domain may be derived from CD28, which gives goodreceptor stability.

Activating Endodomain

The endodomain is the signal-transmission portion of the CAR. Afterantigen recognition, receptors cluster, native CD45 and CD148 areexcluded from the synapse and a signal is transmitted to the cell. Themost commonly used endodomain component is that of CD3-zeta whichcontains 3 ITAMs. This transmits an activation signal to the T cellafter antigen is bound. CD3-zeta may not provide a fully competentactivation signal and additional co-stimulatory signaling may be needed.For example, chimeric CD28 and OX40 can be used with CD3-Zeta totransmit a proliferative/survival signal, or all three can be usedtogether.

In the “OR gate”, the cell of the present invention comprises two CARs,each with an activating endodomain. The activating endodomain is capableof transmitting both immunological signal 1 and immunological signal 2.An endodomain is not considered “activating” if it just comprises CD3ζ,which is sufficient to trigger T-cell killing of cognate target cellsdoes not fully activate the T-cell to proliferate and survive. An“activating endodomain” is a compound endodomains: in which theintracellular part of a T-cell co-stimulatory molecule is fused to thatof CD3ζ resulting in a “second generation” receptor which can transmitan activating and co-stimulatory signal simultaneously after antigenrecognition. The co-stimulatory domain most commonly used is that ofCD28. This supplies the most potent co-stimulatory signal—namelyimmunological signal 2, which triggers T-cell proliferation. In additionto a co-stimulatory domain, the activating endodomain may also includeTNF receptor family endodomains, such as the closely related OX40 and 41BB which transmit survival signals.

An “activating” endodomain.may therefore not comprise the CD3-Zetaendodomain or the CD28 endodomain alone. An activating endodomain may,for example, comprise the CD3-Zeta endodomain with that of either CD28or OX40 or the CD28 endodomain and OX40 and CD3-Zeta endodomain.

Each of the CARs in the OR gate is independently capable of activatingthe T cell. The T cell is thus activated by the presence of eitherantigen alone. The two CARs are not “complementary” in the sense thatactivation of both CARs is necessary to provide activation andco-stimulatory signals.

A endodomain which contains an ITAM motif can act as an activationendodomain in this invention. Several proteins are known to containendodomains with one or more ITAM motifs. Examples of such proteinsinclude the CD3 epsilon chain, the CD3 gamma chain and the CD3 deltachain to name a few. The ITAM motif can be easily recognized as atyrosine separated from a leucine or isoleucine by any two other aminoacids, giving the signature YxxL/I. Typically, but not always, two ofthese motifs are separated by between 6 and 8 amino acids in the tail ofthe molecule (YxxL/Ix(6-8)YxxL/I). Hence, one skilled in the art canreadily find existing proteins which contain one or more ITAM totransmit an activation signal. Further, given the motif is simple and acomplex secondary structure is not required, one skilled in the art candesign polypeptides containing artificial ITAMs to transmit anactivation signal (see WO 2000063372, which relates to syntheticsignalling molecules).

The transmembrane and intracellular T-cell signalling domain(endodomain) of a CAR with an activating endodomain may comprise thesequence shown as SEQ ID No. 16 or 17 or a variant thereof having atleast 80% sequence identity.

comprising CD28 transmembrane domain and CD3 Z endodomain  SEQ ID No. 15FWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRcomprising CD28 transmembrane domain and CD28 and CD3 Zeta endodomains SEQ ID No. 16 FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRcomprising CD28 transmembrane domain and CD28,OX40 and CD3 Zeta endodomains.  SEQ ID No. 17FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity to SEQ ID No. 16 or 17, provided that the sequenceprovides an effective trans-membrane domain and an effectiveintracellular T cell signaling domain.

“Ligation-Off” Inhibitory Endodomain

In the embodiment referred above as the AND gate, one of the CARscomprises an inhibitory endodomain such that the inhibitory CAR inhibitsT-cell activation by the activating CAR in the absence of inhibitory CARligation, but does not significantly inhibit T-cell activation by theactivating CAR when the inhibitory CAR is ligated. This is termed a“ligation-off” inhibitory endodomain.

In this case, the spacer of the inhibitory CAR is of a different length,charge, shape and/or configuration and/or glycosylation from the spacerof the activating CAR, such that when both receptors are ligated, thedifference in spacer dimensions results in isolation of the activatingCARs and the inhibitory CARs in different membrane compartments of theimmunological synapse, so that the activating endodomain is releasedfrom inhibition by the inhibitory endodomain.

The inhibitory endodomains for use in a ligation-off inhibitory CAR maytherefore comprise any sequence which inhibits T-cell signaling by theactivating CAR when it is in the same membrane compartment (i.e. in theabsence of the antigen for the inhibitory CAR) but which does notsignificantly inhibit T cell signaling when it is isolated in a separatepart of the membrane from the inhibitory CAR.

The ligation-off inhibitory endodomain may be or comprise a tyrosinephosphatase, such as a receptor-like tyrosine phosphatase. An inhibitoryendodomain may be or comprise any tyrosine phosphatase that is capableof inhibiting the TCR signalling when only the stimulatory receptor isligated. An inhibitory endodomain may be or comprise any tyrosinephosphatase with a sufficiently fast catalytic rate for phosphorylatedITAMs that is capable of inhibiting the TCR signalling when only thestimulatory receptor is ligated.

For example, the inhibitory endodomain of an AND gate may comprise theendodomain of CD148 or CD45. CD148 and CD45 have been shown to actnaturally on the phosphorylated tyrosines up-stream of TCR signalling.

CD148 is a receptor-like protein tyrosine phosphatase which negativelyregulates TCR signaling by interfering with the phosphorylation andfunction of PLCγ1 and LAT.

CD45 present on all hematopoetic cells, is a protein tyrosinephosphatase which is capable of regulating signal transduction andfunctional responses, again by phosphorylating PLCγ1.

An inhibitory endodomain may comprise all of part of a receptor-liketyrosine phosphatase. The phospatase may interfere with thephosphorylation and/or function of elements involved in T-cellsignalling, such as PLCγ1 and/or LAT.

The transmembrane and endodomain of CD45 and CD148 is shown as SEQ IDNo. 18 and No. 19 respectively.

CD45 trans-membrane and endodomain sequence  SEQ ID 18ALIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGSCD148 trans-membrane and endodomain sequence  SEQ ID 19AVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAP VTTFGKTNGYIA

An inhibitory CAR may comprise all or part of SEQ ID No 18 or 19 (forexample, it may comprise the phosphatase function of the endodomain). Itmay comprise a variant of the sequence or part thereof having at least80% sequence identity, as long as the variant retains the capacity tobasally inhibit T cell signalling by the activating CAR.

Other spacers and endodomains may be tested for example using the modelsystem exemplified herein. Target cell populations can be created bytransducing a suitable cell line such as a SupT1 cell line either singlyor doubly to establish cells negative for both antigens (the wild-type),positive for either and positive for both (e.g. CD19−CD33−, CD19+CD33−,CD19−CD33+ and CD19+CD33+). T cells such as the mouse T cell line BW5147which releases IL-2 upon activation may be transduced with pairs of CARsand their ability to function in a logic gate measured throughmeasurement of IL-2 release (for example by ELISA). For example, it isshown in Example 4 that both CD148 and CD45 endodomains can function asinhibitory CARs in combination with an activating CAR containing a CD3Zeta endodomain. These CARs rely upon a short/non-bulky CD8 stalk spaceron one CAR and a bulky Fc spacer on the other CAR to achieve AND gating.When both receptors are ligated, the difference in spacer dimensionsresults in isolation of the different receptors in different membranecompartments, releasing the CD3 Zeta receptor from inhibition by theCD148 or CD45 endodomains. In this way, activation only occurs once bothreceptors are activated. It can be readily seen that this modular systemcan be used to test alternative spacer pairs and inhibitory endodomains.If the spacers do not achieve isolation following ligation of bothreceptors, the inhibition would not be released and so no activationwould occur. If the inhibitory endodomain under test is ineffective,activation would be expected in the presence of ligation of theactivating CAR irrespective of the ligation status of the inhibitoryCAR.

“Ligation-On” Endodomain

In the embodiment referred above as the AND NOT gate, one of the CARscomprises a “ligation-on” inhibitory endodomain such that the inhibitoryCAR does not significantly inhibit T-cell activation by the activatingCAR in the absence of inhibitory CAR ligation, but inhibits T-cellactivation by the activating CAR when the inhibitory CAR is ligated.

The “ligation-on” inhibitory endodomain may be or comprise a tyrosinephosphatase that is incapable of inhibiting the TCR signalling when onlythe stimulatory receptor is ligated.

The “ligation-on” inhibitory endodomain may be or comprise a tyrosinephosphatase with a sufficiently slow catalytic rate for phosphorylatedITAMs that is incapable of inhibiting the TCR signalling when only thestimulatory receptor is ligated but it is capable of inhibiting the TCRsignalling response when concentrated at the synapse. Concentration atthe synapse is achieved through inhibitory receptor ligation.

If a tyrosine phosphatase has a catalytic rate which is too fast for a“ligation-on” inhibitory endodomain, then it is possible to tune-downthe catalytic rates of phosphatase through modification such as pointmutations and short linkers (which cause steric hindrance) to make itsuitable for a “ligation-on” inhibitory endodomain.

In this first embodiment the endodomain may be or comprise a phosphatasewhich is considerably less active than CD45 or CD148, such thatsignificant dephosphorylation of ITAMS only occurs when activating andinhibitory endodomains are co-localised. Many suitable sequences areknown in the art. For example, the inhibitory endodomain of a NOT ANDgate may comprise all or part of a protein-tyrosine phosphatase such asPTPN6.

Protein tyrosine phosphatases (PTPs) are signaling molecules thatregulate a variety of cellular processes including cell growth,differentiation, mitotic cycle, and oncogenic transformation. TheN-terminal part of this PTP contains two tandem Src homolog (SH2)domains, which act as protein phospho-tyrosine binding domains, andmediate the interaction of this PTP with its substrates. This PTP isexpressed primarily in hematopoietic cells, and functions as animportant regulator of multiple signaling pathways in hematopoieticcells.

The inhibitor domain may comprise all of PTPN6 (SEQ ID No. 20) or justthe phosphatase domain (SEQ ID No. 21).

sequence of PTPN6  SEQ ID 20MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRKsequence of phosphatase domain of PTPN6  SEQ ID 21FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVINCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQF

A second embodiment of a ligation-on inhibitory endodomain is an ITIM(Immunoreceptor Tyrosine-based Inhibition motif) containing endodomainsuch as that from CD22, LAIR-1, the Killer inhibitory receptor family(KIR), LILRB1, CTLA4, PD-1, BTLA etc. When phosphorylated, ITIMsrecruits endogenous PTPN6 through its SH2 domain. If co-localised withan ITAM containing endodomain, dephosphorylation occurs and theactivating CAR is inhibited.

An ITIM is a conserved sequence of amino acids (S/I/V/LxYxxI/V/L) thatis found in the cytoplasmic tails of many inhibitory receptors of theimmune system. One skilled in the art can easily find protein domainscontaining an ITIM. A list of human candidate ITIM-containing proteinshas been generated by proteome-wide scans (Staub, et al (2004) Cell.Signal. 16, 435-456). Further, since the consensus sequence is wellknown and little secondary structure appears to be required, one skilledin the art could generate an artificial ITIM.

ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1,KIR2DL4, KIR2DL5, KIR3DL1 and KIR3DL3 are shown in SEQ ID 22 to 31respectively

PDCD1 endodomain SEQ ID 22CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL BTLA4 SEQ ID 23KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH LILRB1 SEQ ID 24LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPS QEGPSPAVPSIYATLAIHLAIR1 SEQ ID 25 HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVAR H CTLA4 SEQ ID 26FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPEC EKQFQPYFIPIN KIR2DL1SEQ ID 27 GNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPN AESRSKVVSCP KIR2DL4SEQ ID 28 GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRTVNREDSDEQDPQEVTYAQLDHCIFTQRKITGPSQRSKRPSTDTSVCIELPNAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPAAGI KIR2DL5 SEQ ID 29TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLDHCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPAAGI KIR3DL1 SEQ ID 30KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTANSEDSDEQDPEEVTYAQLDHCVFTQRKITRPSQRPKTPPTDTILYTELPN AKPRSKVVSCP KIR3DL3SEQ ID 31 KDPGNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYAQLNHCVFTQRKITRPSQRPKTPPTDTSV

A third embodiment of a ligation-on inhibitory endodomain is an ITIMcontaining endodomain co-expressed with a fusion protein. The fusionprotein may comprise at least part of a protein-tyrosine phosphatase andat least part of a receptor-like tyrosine phosphatase. The fusion maycomprise one or more SH2 domains from the protein-tyrosine phosphatase.For example, the fusion may be between a PTPN6 SH2 domain and CD45endodomain or between a PTPN6 SH2 domain and CD148 endodomain. Whenphosphorylated, the ITIM domains recruit the fusion protein bring thehighly potent CD45 or CD148 phosphatase to proximity to the activatingendodomain blocking activation.

Sequences of fusion proteins are listed 32 and 33

PTPN6-CD45 fusion protein SEQ ID 32WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYKIYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLKETIGDFMIQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPAS PALNQGSPTPN6-CD148 fusion SEQ ID 33ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA

A ligation-on inhibitory CAR may comprise all or part of SEQ ID No 20 or21. It may comprise all or part of SEQ ID 22 to 31. It may comprise allor part of SEQ ID 22 to 31 co-expressed with either SEQ ID 32 or 33. Itmay comprise a variant of the sequence or part thereof having at least80% sequence identity, as long as the variant retains the capacity toinhibit T cell signaling by the activating CAR upon ligation of theinhibitory CAR.

As above, alternative spacers and endodomains may be tested for exampleusing the model system exemplified herein. It is shown in Example 5 thatthe PTPN6 endodomain can function as a semi-inhibitory CAR incombination with an activating CAR containing a CD3 Zeta endodomain.These CARs rely upon a human CD8 stalk spacer on one CAR and a mouse CD8stalk spacer on the other CAR. The orthologous sequences prevent crosspairing. However, when both receptors are ligated, the similaritybetween the spacers results in co-segregation of the different receptorsin the same membrane compartments. This results in inhibition of the CD3Zeta receptor by the PTPN6 endodomain. If only the activating CAR isligated the PTPN6 endodomain is not sufficiently active to prevent Tcell activation. In this way, activation only occurs if the activatingCAR is ligated and the inhibitory CAR is not ligated (AND NOT gating).It can be readily seen that this modular system can be used to testalternative spacer pairs and inhibitory domains. If the spacers do notachieve co-segregation following ligation of both receptors, theinhibition would not be effective and so activation would occur. If thesemi-inhibitory endodomain under test is ineffective, activation wouldbe expected in the presence of ligation of the activating CARirrespective of the ligation status of the semi-inhibitory CAR.

Co-Expression Site

The second aspect of the invention relates to a nucleic acid whichencodes the first and second CARs.

The nucleic acid may produce a polypeptide which comprises the two CARmolecules joined by a cleavage site. The cleavage site may beself-cleaving, such that when the polypeptide is produced, it isimmediately cleaved into the first and second CARs without the need forany external cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouthdisease virus (FMDV) 2a self-cleaving peptide, which has the sequenceshown as SEQ ID No. 34:

SEQ ID No. 34  RAEGRGSLLTCGDVEENPGP. 

The co-expressing sequence may be an internal ribosome entry sequence(IRES). The co-expressing sequence may be an internal promoter.

Cell

The first aspect of the invention relates to a cell which co-expresses afirst CAR and a second CAR at the cell surface.

The cell may be any eukaryotic cell capable of expressing a CAR at thecell surface, such as an immunological cell.

In particular the cell may be an immune effector cell such as a T cellor a natural killer (NK) cell.

T cells or T lymphocytes are a type of lymphocyte that play a centralrole in cell-mediated immunity. They can be distinguished from otherlymphocytes, such as B cells and natural killer cells (NK cells), by thepresence of a T-cell receptor (TCR) on the cell surface. There arevarious types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells inimmunologic processes, including maturation of B cells into plasma cellsand memory B cells, and activation of cytotoxic T cells and macrophages.TH cells express CD4 on their surface. TH cells become activated whenthey are presented with peptide antigens by MHC class II molecules onthe surface of antigen presenting cells (APCs). These cells candifferentiate into one of several subtypes, including TH1, TH2, TH3,TH17, Th9, or TFH, which secrete different cytokines to facilitatedifferent types of immune responses.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. CTLsexpress the CD8 at their surface. These cells recognize their targets bybinding to antigen associated with MHC class I, which is present on thesurface of all nucleated cells. Through IL-10, adenosine and othermolecules secreted by regulatory T cells, the CD8+ cells can beinactivated to an anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise three subtypes: central memory T cells (TCMcells) and two types of effector memory T cells (TEM cells and TEMRAcells). Memory cells may be either CD4+ or CD8+. Memory T cellstypically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described—naturallyoccurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Tregcells) arise in the thymus and have been linked to interactions betweendeveloping T cells with both myeloid (CD11c+) and plasmacytoid (CD123+)dendritic cells that have been activated with TSLP. Naturally occurringTreg cells can be distinguished from other T cells by the presence of anintracellular molecule called FoxP3. Mutations of the FOXP3 gene canprevent regulatory T cell development, causing the fatal autoimmunedisease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originateduring a normal immune response.

The T cell of the invention may be any of the T cell types mentionedabove, in particular a CTL.

Natural killer (NK) cells are a type of cytolytic cell which forms partof the innate immune system. NK cells provide rapid responses to innatesignals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are definedas large granular lymphocytes (LGL) and constitute the third kind ofcells differentiated from the common lymphoid progenitor generating Band T lymphocytes. NK cells are known to differentiate and mature in thebone marrow, lymph node, spleen, tonsils and thymus where they thenenter into the circulation.

The CAR cells of the invention may be any of the cell types mentionedabove.

CAR− expressing cells, such as CAR-expressing T or NK cells may eitherbe created ex vivo either from a patient's own peripheral blood (1stparty), or in the setting of a haematopoietic stem cell transplant fromdonor peripheral blood (2^(nd) party), or peripheral blood from anunconnected donor (3^(rd) party).

The present invention also provide a cell composition comprising CARexpressing T cells and/or CAR expressing NK cells according to thepresent invention. The cell composition may be made by tranducing ablood-sample ex vivo with a nucleic acid according to the presentinvention.

Alternatively, CAR-expressing cells may be derived from ex vivodifferentiation of inducible progenitor cells or embryonic progenitorcells to the relevant cell type, such as T cells. Alternatively, animmortalized cell line such as a T-cell line which retains its lyticfunction and could act as a therapeutic may be used.

In all these embodiments, CAR cells are generated by introducing DNA orRNA coding for the CARs by one of many means including transduction witha viral vector, transfection with DNA or RNA.

A CAR T cell of the invention may be an ex vivo T cell from a subject.The T cell may be from a peripheral blood mononuclear cell (PBMC)sample. T cells may be activated and/or expanded prior to beingtransduced with CAR-encoding nucleic acid, for example by treatment withan anti-CD3 monoclonal antibody.

A CAR T cell of the invention may be made by:

-   -   (i) isolation of a T cell-containing sample from a subject or        other sources listed above; and    -   (ii) transduction or transfection of the T cells with one or        more nucleic acid sequence(s) encoding the first and second CAR.

The T cells may then by purified, for example, selected on the basis ofco-expression of the first and second CAR.

Nucleic Acid Sequences

The second aspect of the invention relates to one or more nucleic acidsequence(s) which codes for a first CAR and a second CAR as defined inthe first aspect of the invention.

The nucleic acid sequence may comprise one of the following sequences,or a variant thereof

SEQ ID 35 OR gate

SEQ ID 36 AND gate using CD45

SEQ ID 37 AND gate using CD148

SEQ ID 38 AND NOT gate using PTPN6 as endodomain

SEQ ID 39 AND NOT gate using LAIR1 endodomain

SEQ ID 40 AND NOT gate using LAIR1 and PTPN6 SH2 fusion with CD148phosphatase

SEQ ID No. 35: >MP13974.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-CD28tmZwATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGTTCTGGGTCCTGGTGGTGGTGGGAGGCGTGCTGGCCTGTTACTCTCTCCTGGTGACCGTGGCCTTCATCATCTTTTGGGTGCGCTCCCGGGTGAAGTTTTCTCGCTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAATGAACTGAACCTGGGCAGGCGGGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGACCCCGAGATGGGCGGCAAACCACGGCGCAAAAATCCCCAGGAGGGACTCTATAACGAGCTGCAGAAGGACAAAATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGAAGACGCGGAAAGGGCCACGACGGCCTGTATCAGGGATTGTCCACCGCTACAAAAGATACATATGATGCCCTGCACATGCAGGCCCTGCCACCCAGATGASEQ ID No. 36 >MP14802.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-dCD45ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCACTGATAGCATTTCTGGCATTTCTGATTATTGTGACATCAATAGCCCTGCTTGTTGTTCTCTACAAAATCTATGATCTACATAAGAAAAGATCCTGCAATTTAGATGAACAGCAGGAGCTTGTTGAAAGGGATGATGAAAAACAACTGATGAATGTGGAGCCAATCCATGCAGATATTTTGTTGGAAACTTATAAGAGGAAGATTGCTGATGAAGGAAGACTTTTTCTGGCTGAATTTCAGAGCATCCCGCGGGTGTTCAGCAAGTTTCCTATAAAGGAAGCTCGAAAGCCCTTTAACCAGAATAAAAACCGTTATGTTGACATTCTTCCTTATGATTATAACCGTGTTGAACTCTCTGAGATAAACGGAGATGCAGGGTCAAACTACATAAATGCCAGCTATATTGATGGTTTCAAAGAACCCAGGAAATACATTGCTGCACAAGGTCCCAGGGATGAAACTGTTGATGATTTCTGGAGGATGATTTGGGAACAGAAAGCCACAGTTATTGTCATGGTCACTCGATGTGAAGAAGGAAACAGGAACAAGTGTGCAGAATACTGGCCGTCAATGGAAGAGGGCACTCGGGCTTTTGGAGATGTTGTTGTAAAGATCAACCAGCACAAAAGATGTCCAGATTACATCATTCAGAAATTGAACATTGTAAATAAAAAAGAAAAAGCAACTGGAAGAGAGGTGACTCACATTCAGTTCACCAGCTGGCCAGACCACGGGGTGCCTGAGGATCCTCACTTGCTCCTCAAACTGAGAAGGAGAGTGAATGCCTTCAGCAATTTCTTCAGTGGTCCCATTGTGGTGCACTGCAGTGCTGGTGTTGGGCGCACAGGAACCTATATCGGAATTGATGCCATGCTAGAAGGCCTGGAAGCCGAGAACAAAGTGGATGTTTATGGTTATGTTGTCAAGCTAAGGCGACAGAGATGCCTGATGGTTCAAGTAGAGGCCCAGTACATCTTGATCCATCAGGCTTTGGTGGAATACAATCAGTTTGGAGAAACAGAAGTGAATTTGTCTGAATTACATCCATATCTACATAACATGAAGAAAAGGGATCCACCCAGTGAGCCGTCTCCACTAGAGGCTGAATTCCAGAGACTTCCTTCATATAGGAGCTGGAGGACACAGCACATTGGAAATCAAGAAGAAAATAAAAGTAAAAACAGGAATTCTAATGTCATCCCATATGACTATAACAGAGTGCCACTTAAACATGAGCTGGAAATGAGTAAAGAGAGTGAGCATGATTCAGATGAATCCTCTGATGATGACAGTGATTCAGAGGAACCAAGCAAATACATCAATGCATCTTTTATAATGAGCTACTGGAAACCTGAAGTGATGATTGCTGCTCAGGGACCACTGAAGGAGACCATTGGTGACTTTTGGCAGATGATCTTCCAAAGAAAAGTCAAAGTTATTGTTATGCTGACAGAACTGAAACATGGAGACCAGGAAATCTGTGCTCAGTACTGGGGAGAAGGAAAGCAAACATATGGAGATATTGAAGTTGACCTGAAAGACACAGACAAATCTTCAACTTATACCCTTCGTGTCTTTGAACTGAGACATTCCAAGAGGAAAGACTCTCGAACTGTGTACCAGTACCAATATACAAACTGGAGTGTGGAGCAGCTTCCTGCAGAACCCAAGGAATTAATCTCTATGATTCAGGTCGTCAAACAAAAACTTCCCCAGAAGAATTCCTCTGAAGGGAACAAGCATCACAAGAGTACACCTCTACTCATTCACTGCAGGGATGGATCTCAGCAAACGGGAATATTTTGTGCTTTGTTAAATCTCTTAGAAAGTGCGGAAACAGAAGAGGTAGTGGATATTTTTCAAGTGGTAAAAGCTCTACGCAAAGCTAGGCCAGGCATGGTTTCCACATTCGAGCAATATCAATTCCTATATGACGTCATTGCCAGCACCTACCCTGCTCAGAATGGACAAGTAAAGAAAAACAACCATCAAGAAGATAAAATTGAATTTGATAATGAAGTGGACAAAGTAAAGCAGGATGCTAATTGTGTTAATCCACTTGGTGCCCCAGAAAAGCTCCCTGAAGCAAAGGAACAGGCTGAAGGTTCTGAACCCACGAGTGGCACTGAGGGGCCAGAACATTCTGTCAATGGTCCTGCAAGTCCAGCTTTAAATCAAGGTTCATAGSEQ ID No. 37: >MP14801.SFG.aCD19fmc63_clean-CD8STK-CD28tmZ-2A-aCD33glx-HCH2CH3pvaa-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAASEQ ID No. 38 >16076.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-tm-dPTPN6ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATCTACTGGGCACCTCTGGCCGGAATATGCGTGGCACTGCTGCTGAGCCTCATCATCACCCTGATCTGTTATCACCGAAGCCGCAAGCGGGTGTGTAAAAGTGGAGGCGGAAGCTTCTGGGAGGAGTTTGAGAGTTTGCAGAAGCAGGAGGTGAAGAACTTGCACCAGCGTCTGGAAGGGCAGCGGCCAGAGAACAAGGGCAAGAACCGCTACAAGAACATTCTCCCCTTTGACCACAGCCGAGTGATCCTGCAGGGACGGGACAGTAACATCCCCGGGTCCGACTACATCAATGCCAACTACATCAAGAACCAGCTGCTAGGCCCTGATGAGAACGCTAAGACCTACATCGCCAGCCAGGGCTGTCTGGAGGCCACGGTCAATGACTTCTGGCAGATGGCGTGGCAGGAGAACAGCCGTGTCATCGTCATGACCACCCGAGAGGTGGAGAAAGGCCGGAACAAATGCGTCCCATACTGGCCCGAGGTGGGCATGCAGCGTGCTTATGGGCCCTACTCTGTGACCAACTGCGGGGAGCATGACACAACCGAATACAAACTCCGTACCTTACAGGTCTCCCCGCTGGACAATGGAGACCTGATTCGGGAGATCTGGCATTACCAGTACCTGAGCTGGCCCGACCACGGGGTCCCCAGTGAGCCTGGGGGTGTCCTCAGCTTCCTGGACCAGATCAACCAGCGGCAGGAAAGTCTGCCTCACGCAGGGCCCATCATCGTGCACTGCAGCGCCGGCATCGGCCGCACAGGCACCATCATTGTCATCGACATGCTCATGGAGAACATCTCCACCAAGGGCCTGGACTGTGACATTGACATCCAGAAGACCATCCAGATGGTGCGGGCGCAGCGCTCGGGCATGGTGCAGACGGAGGCGCAGTACAAGTTCATCTACGTGGCCATCGCCCAGTTCATTGAAACCACTAAGAAGAAGCTGTGASEQ ID No. 39 >MP16091.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-endoATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACTGASEQ ID no. 40 >MP16092.SFG.aCD19fmc63-CD8STK-CD28tmZ-2A-aCD33glx-muCD8STK-LAIR1tm-endo-2A-PTPN6_SH2-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGCCGTGCCCACTCAGGTCCTGGGGTTGTTGCTACTGTGGCTTACAGATGCCAGATGTGACATCCAGATGACACAGTCTCCATCTTCCCTGTCTGCATCTGTCGGAGATCGCGTCACCATCACCTGTCGAGCAAGTGAGGACATTTATTTTAATTTAGTGTGGTATCAGCAGAAACCAGGAAAGGCCCCTAAGCTCCTGATCTATGATACAAATCGCTTGGCAGATGGGGTCCCATCACGGTTCAGTGGCTCTGGATCTGGCACACAGTATACTCTAACCATAAGTAGCCTGCAACCCGAAGATTTCGCAACCTATTATTGTCAACACTATAAGAATTATCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAAATCAAAAGATCTGGTGGCGGAGGGTCAGGAGGCGGAGGCAGCGGAGGCGGTGGCTCGGGAGGCGGAGGCTCGAGATCTGAGGTGCAGTTGGTGGAGTCTGGGGGCGGCTTGGTGCAGCCTGGAGGGTCCCTGAGGCTCTCCTGTGCAGCCTCAGGATTCACTCTCAGTAATTATGGCATGCACTGGATCAGGCAGGCTCCAGGGAAGGGTCTGGAGTGGGTCTCGTCTATTAGTCTTAATGGTGGTAGCACTTACTATCGAGACTCCGTGAAGGGCCGATTCACTATCTCCAGGGACAATGCAAAAAGCACCCTCTACCTTCAAATGAATAGTCTGAGGGCCGAGGACACGGCCGTCTATTACTGTGCAGCACAGGACGCTTATACGGGAGGTTACTTTGATTACTGGGGCCAAGGAACGCTGGTCACAGTCTCGTCTATGGATCCCGCCACCACAACCAAGCCCGTGCTGCGGACCCCAAGCCCTGTGCACCCTACCGGCACCAGCCAGCCTCAGAGACCCGAGGACTGCCGGCCTCGGGGCAGCGTGAAGGGCACCGGCCTGGACTTCGCCTGCGACATTCTCATCGGGGTCTCAGTGGTCTTCCTCTTCTGTCTCCTCCTCCTGGTCCTCTTCTGCCTCCATCGCCAGAATCAGATAAAGCAGGGGCCCCCCAGAAGCAAGGACGAGGAGCAGAAGCCACAGCAGAGGCCTGACCTGGCTGTTGATGTTCTAGAGAGGACAGCAGACAAGGCCACAGTCAATGGACTTCCTGAGAAGGACCGGGAGACCGACACCAGCGCCCTGGCTGCAGGGAGTTCCCAGGAGGTGACGTATGCTCAGCTGGACCACTGGGCCCTCACACAGAGGACAGCCCGGGCTGTGTCCCCACAGTCCACAAAGCCCATGGCCGAGTCCATCACGTATGCAGCCGTTGCCAGACACAGGGCAGAAGGAAGAGGTAGCCTGCTGACTTGCGGGGACGTGGAAGAGAACCCAGGGCCATGGTATCATGGCCACATGTCTGGCGGGCAGGCAGAGACGCTGCTGCAGGCCAAGGGCGAGCCCTGGACGTTTCTTGTGCGTGAGAGCCTCAGCCAGCCTGGAGACTTCGTGCTTTCTGTGCTCAGTGACCAGCCCAAGGCTGGCCCAGGCTCCCCGCTCAGGGTCACCCACATCAAGGTCATGTGCGAGGGTGGACGCTACACAGTGGGTGGTTTGGAGACCTTCGACAGCCTCACGGACCTGGTGGAGCATTTCAAGAAGACGGGGATTGAGGAGGCCTCAGGCGCCTTTGTCTACCTGCGGCAGCCGTACAGCGGTGGCGGTGGCAGCTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCAGCGGTAGCTAA

The nucleic acid sequence may encode the same amino acid sequence asthat encoded by SEQ ID No. 35, 36, 37, 38, 39 or 40, but may have adifferent nucleic acid sequence, due to the degeneracy of the geneticcode. The nucleic acid sequence may have at least 80, 85, 90, 95, 98 or99% identity to the sequence shown as SEQ ID No. 35, 36, 37, 38, 39 or40, provided that it encodes a first CAR and a second CAR as defined inthe first aspect of the invention.

Vector

The present invention also provides a vector, or kit of vectors whichcomprises one or more CAR-encoding nucleic acid sequence(s). Such avector may be used to introduce the nucleic acid sequence(s) into a hostcell so that it expresses the first and second CARs.

The vector may, for example, be a plasmid or a viral vector, such as aretroviral vector or a lentiviral vector, or a transposon based vectoror synthetic mRNA.

The vector may be capable of transfecting or transducing a T cell.

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a plurality of CAR-expressing T cells or NK cells accordingto the first aspect of the invention. The pharmaceutical composition mayadditionally comprise a pharmaceutically acceptable carrier, diluent orexcipient. The pharmaceutical composition may optionally comprise one ormore further pharmaceutically active polypeptides and/or compounds. Sucha formulation may, for example, be in a form suitable for intravenousinfusion.

Method of Treatment

The T cells of the present invention may be capable of killing targetcells, such as cancer cells. The target cell may be recognisable by adefined pattern of antigen expression, for example the expression ofantigen A AND antigen B; the expression of antigen A OR antigen B; orthe expression of antigen A AND NOT antigen B or complex iterations ofthese gates.

T cells of the present invention may be used for the treatment of aninfection, such as a viral infection.

T cells of the invention may also be used for the control of pathogenicimmune responses, for example in autoimmune diseases, allergies andgraft-vs-host rejection.

T cells of the invention may be used for the treatment of a cancerousdisease, such as bladder cancer, breast cancer, colon cancer,endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer,melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer andthyroid cancer.

It is particularly suited for treatment of solid tumours where theavailability of good selective single targets is limited.

T cells of the invention may be used to treat: cancers of the oralcavity and pharynx which includes cancer of the tongue, mouth andpharynx; cancers of the digestive system which includes oesophageal,gastric and colorectal cancers; cancers of the liver and biliary treewhich includes hepatocellular carcinomas and cholangiocarcinomas;cancers of the respiratory system which includes bronchogenic cancersand cancers of the larynx; cancers of bone and joints which includesosteosarcoma; cancers of the skin which includes melanoma; breastcancer; cancers of the genital tract which include uterine, ovarian andcervical cancer in women, prostate and testicular cancer in men; cancersof the renal tract which include renal cell carcinoma and transitionalcell carcinomas of the utterers or bladder; brain cancers includinggliomas, glioblastoma multiforme and medullobastomas; cancers of theendocrine system including thyroid cancer, adrenal carcinoma and cancersassociated with multiple endocrine neoplasm syndromes; lymphomasincluding Hodgkin's lymphoma and non-Hodgkin lymphoma; Multiple Myelomaand plasmacytomas; leukaemias both acute and chronic, myeloid orlymphoid; and cancers of other and unspecified sites includingneuroblastoma.

Treatment with the T cells of the invention may help prevent the escapeor release of tumour cells which often occurs with standard approaches.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

EXAMPLES Example 1—Creation of Target Cell Populations

For the purposes of proving the principle of the invention, receptorsbased on anti-CD19 and anti-CD33 were arbitrarily chosen. Usingretroviral vectors, CD19 and CD33 were cloned. These proteins weretruncated so that they do not signal and could be stably expressed forprolonged periods. Next, these vectors were used to transduce the SupT1cell line either singly or doubly to establish cells negative for bothantigen (the wild-type), positive for either and positive for both. Theexpression data are shown in FIG. 3.

Example 2—Design and Function of the OR Gate

To construct the OR gate, a pair of receptors recognizing CD19 and CD33were co-expressed. Different spacers were used to prevent cross-pairing.Both receptors had a trans-membrane domain derived from CD28 to improvesurface stability and an endodomain derived from that of CD3 Zeta toprovide a simple activating signal. In this way, a pair of independent1st generation CARs were co-expressed. The retroviral vector cassetteused to co-express the sequences utilizes a foot-and-mouth 2Aself-cleaving peptide to allow co-expression 1:1 of both receptors. Thecassette design is shown in FIG. 4, and the protein structures in FIG.5. The nucleotide sequence of homologous regions was codon-wobbled toprevent recombination during retroviral vector reverse transcription.

Example 3—Testing the OR Gate

Expression of both CARs was tested on the T-cell surface by stainingwith cognate antigen fused to Fc. By using different species of Fcdomains (mouse for CD19 and rabbit for CD33), co-expression of both CARswas determined on the cell surface by staining with different secondaryantibodies conjugated with different fluorophores. This is shown in FIG.6.

Functional testing was then carried out using the mouse T-cell lineBW5147. This cell line releases IL2 upon activation allowing a simplequantitative readout. These T-cells were co-cultured with increasingamounts of the artificial target cells described above. T-cellsresponded to target cells expressing either antigen, as shown by IL2release measured by ELISA. Both CARs were shown to be expressed on thecell surfaces and the T-cells were shown to respond to either or bothantigens. These data are show in FIG. 7.

Example 4—Design and Function of the AND Gate

The AND gate combines a simple activating receptor with a receptor whichbasally inhibits activity, but whose inhibition is turned off once thereceptor is ligated. This was achieved by combining a standard 1Stgeneration CAR with a short/non-bulky CD8 stalk spacer and a CD3 Zetaendodomain with a second receptor with a bulky Fc spacer whoseendodomain contained either CD148 or CD45 endodomains. When bothreceptors are ligated, the difference in spacer dimensions results inisolation of the different receptors in different membrane compartments,releasing the CD3 Zeta receptor from inhibition by the CD148 or CD45endodomains. In this way, activation only occurs once both receptors areactivated. CD148 and CD45 were chosen for this as they function in thismanner natively: for instance, the very bulky CD45 ectodomain excludesthe entire receptor from the immunological synapse. The expressioncassette is depicted in FIG. 8 and the subsequent proteins in FIG. 9.

Surface staining for the different specificity showed that both receptorpairs could be effectively expressed on the cell surface shown in FIG.10. Function in BW5147 shows that the T-cell is only activated in thepresence of both antigens (FIG. 11).

Example 5: Demonstration of Generalizability of the AND Gate

To ensure that the observations were not a manifestation of somespecific characteristic of CD19/CD33 and their binders which had beenused, the two targeting scFvs were swapped such that now, the activation(ITAM) signal was transmitted upon recognition of CD33, rather thanCD19; and the inhibitory (CD148) signal was transmitted upon recognitionof CD19, rather than of CD33. Since CD45 and CD148 endodomains areconsidered to be functionally similar, experimentation was restricted toAND gates with CD148 endodomain. This should still result in afunctional AND gate. T-cells expressing the new logic gate wherechallenged with targets bearing either CD19 or CD33 alone, or both. TheT-cells responded to targets expressing both CD19 and CD33, but not totargets expressing only one or none of these antigens. This shows thatthe AND gate is still functional in this format (FIG. 18B).

On the same lines, it was sought to establish how generalizable our ANDgate is: the AND gate should be generalizable across different targets.While there may be lesser or greater fidelity of the gate given relativeantigen density, cognate scFv binding kinetics and precise distance ofthe scFv binding epitope, one would expect to see some AND gatemanifestations with a wide set of targets and binders. To test this,three additional AND gates were generated. Once again, experimentationwas restricted to the CD148 version of the AND gate. The second scFvfrom the original CD148 AND gate was replaced with the anti-GD2 scFvhuK666 (SEQ ID 41 and SEQ ID 42), or with the anti-CD5 scFv (SEQ ID 43and SEQ ID 44), or the anti-EGFRvIII scFv MR1.1 (SEQ ID 45 AND SEQ ID46) to generate the following CAR AND gates: CD19 AND GD2; CD19 AND CD5;CD19 AND EGFRvIII. The following artificial antigen expressing celllines were also generated: by transducing SupT1, and our SupT1.CD19 withGM3 and GD2 synthases SupT1.GD2 and SupT1.CD19.GD2 were generated. Bytransducing SupT1 and SupT1.CD19 with a retroviral vector coding forEGFRvIII SupT1.EGFRvIII and SupT1.CD19.EGFRvIII were generated. SinceCD5 is expressed on SupT1 cells, a different cell line was used togenerate the target cells: 293T cells were generated which express CD19alone, CD5 alone and both CD5 and CD19 together. Expression wasconfirmed by flow-cytometry (FIG. 19). T-cells expressing the three newCAR AND gates were challenged with SupT1.CD19 and respective cognatedouble positive and single positive target cells. All three AND gatesdemonstrated reduced activation by the double positive cell lines incomparison with the single positive targets (FIG. 20). This demonstratesgeneralizability of the AND gate design to arbitrary targets and cognatebinders.

Example 6: Experimental Proof of Kinetic Segregation Model of CAR ANDGate

The aim was to prove the model that differential segregation caused bydifferent spacers is the central mechanism behind the ability togenerate these logic CAR gates. The model is that if only the activatingCAR is ligated, the potent inhibiting ‘ligation off’ type CAR is insolution in the membrane and can inhibit the activating CAR. Once bothCARs are ligated, if both CAR spacers are sufficiently different, theywill segregate within the synapse and not co-localize. Hence, a keyrequirement is that the spacers are sufficiently different. If the modelis correct, if both spacers are sufficiently similar so they co-localizewhen both receptors are ligated, the gate will fail to function. To testthis, the “bulky” Fc spacer in the original CAR we replaced with amurine CD8 spacer. It was predicted that this has the similar length,bulk and charge as human CD8 but so should not cross-pair with it.Hence, the new gate had a first CAR which recognizes CD19, a human CD8stalk spacer and an activatory endodomain; while the second CARrecognizes CD33, has a mouse CD8 stalk spacer and a CD148 endodomain(FIG. 18C). T-cells were transduced to express this new CAR gate. TheseT-cells were then challenged with SupT1 cells expressing CD19 alone,CD33 alone or CD19 and CD33 together. T-cells did not respond to SupT1cells expressing either antigen alone as per the original AND gate.However, CAR T-cells failed to respond to SupT1 cells expressing bothantigens, thereby confirming the model (FIG. 18C). A functional AND gaterequires both CARs to have spacers sufficiently different so that theydo not co-localize within an immunological synapse (FIGS. 23A and B).

Example 7—Design and Function of an AND NOT Gate

Phosphatases such as CD45 and CD148 are so potent that even a smallamount entering an immunological synapse can inhibit ITAM activation.This is the basis of inhibition of the logical AND gate. Other classesof phosphatases are not as potent e.g. PTPN6 and related phosphatases.It was predicted that a small amount of PTPN6 entering a synapse bydiffusion would not inhibit activation. In addition, it was predictedthat if an inhibitory CAR had a sufficiently similar spacer to anactivating CAR, it could co-localize within a synapse if both CARs wereligated. In this case, large amounts of the inhibitory endodomain wouldbe sufficient to stop the ITAMS from activating when both antigens werepresent. In this way, an AND NOT gate could be created.

For the NOT AND gate, the second signal needs to “veto” activation. Thisis done by bringing an inhibitory signal into the immunological synapse,for example by bringing in the phosphatase of an enzyme such as PTPN6.We hence generated an initial AND NOT gate as follows: two CARsco-expressed whereby the first recognizes CD19, has a human CD8 stalkspacer and an activating endodomain; co-expressed with an anti-CD33 CARwith a mouse CD8 stalk spacer and an endodomain comprising of thecatalytic domain of PTPN6 (SEQ ID 38, FIG. 13 A with B). A suitablecassette is shown in FIG. 12 and preliminary functional data are shownin FIG. 14.

In addition, an alternative strategy was developed for generating an ANDNOT gate. Immune Tyrosinase Inhibitory Motifs (ITIMs) are activated in asimilar manner to ITAMS, in that they become phosphorylated by Ick uponclustering and exclusion of phosphatases. Instead of triggeringactivation by binding ZAP70, phosphorylated ITIMs recruit phosphataseslike PTPN6 through their cognate SH2 domains. An ITIM can function as aninhibitory endodomain, as long as the spacers on the activating andinhibiting CARs can co-localize. To generate this construct, an AND NOTgate was generated as follows: two CARs co-expressed—the firstrecognizes CD19, has a human CD8 stalk spacer and an activatingendodomain; co-expressed with an anti-CD33 CAR with a mouse CD8 stalkspacer and an ITIM containing endodomain derived from that of LAIR1 (SEQID 39, FIG. 13 A with C).

A further, more complex AND NOT gate was also developed, whereby an ITIMis enhanced by the presence of an additional chimeric protein: anintracellular fusion of the SH2 domain of PTPN6 and the endodomain ofCD148. In this design three proteins are expressed—the first recognizesCD19, has a human CD8 stalk spacer and an activating endodomain;co-expressed with an anti-CD33 CAR with a mouse CD8 stalk spacer and anITIM containing endodomain derived from that of LAIR1. A further 2Apeptide, allows co-expression of the PTPN6-CD148 fusion (SEQ ID 40,FIGS. 13 A and D). It was predicted that these AND NOT gates would havea different range of inhibition: PTPN6-CD148>PTPN6>>ITIM.

T-cells were transduced with these gates and challenged with targetsexpressing either CD19 or CD33 alone, or both CD19 and CD33 together.All three gates responded to targets expressing only CD19, but nottargets expressing both CD19 and CD33 together (FIG. 21), confirmingthat all three of the AND NOT gates were functional.

Example 8: Experimental Proof of Kinetic Segregation Model of PTPN6Based AND NOT Gate

The model of the AND NOT gate centres around the fact that the nature ofthe spacers used in both CARs is pivotal for the correct function of thegate. In the functional AND NOT gate with PTPN6, both CAR spacers aresufficiently similar that when both CARs are ligated, both co-localizewithin the synapse so the high concentration even the weak PTPN6 issufficient to inhibit activation. If the spacers were different,segregation in the synapse will isolate the PTPN6 from the ITAM allowingactivation disrupting the AND NOT gate. To test this, a control wasgenerated replacing the murine CD8 stalk spacer with that of Fc. In thiscase, the test gate consisted of two CARs, the first recognizes CD19,has a human CD8 stalk spacer and an ITAM endodomain; while the secondCAR recognizes CD33, has an Fc spacer and an endodomain comprising ofthe phosphatase from PTPN6. This gate activates in response to CD19, butalso activates in response to CD19 and CD33 together (FIG. 22B, wherefunction of this gate is compared with that of the original AND NOT, andthe control AND gate variant described in Example 6). This experimentaldata proves the model that for a functional AND NOT gate with PTPN6,co-localizing spacers are needed.

Example 9: Experimental Proof of Kinetic Segregation Model of ITIM BasedAND NOT Gate

Similar to the PTPN6 based AND NOT gate, the ITIM based gate alsorequires co-localization in an immunological synapse to function as anAND NOT gate. To prove this hypothesis, a control ITIM based gate wasgenerated as follows: two CARs co-expressed—the first recognizes CD19,has a human CD8 stalk spacer and an activating endodomain; co-expressedwith an anti-CD33 CAR with an Fc spacer and an ITIM containingendodomain derived from that of LAIR1. The activity of this gate wascompared with that of the original ITIM based AND NOT gate. In thiscase, the modified gate activated in response to targets expressingCD19, but also activated in response to cells expressing both CD19 andCD33. These data indicate that ITIM based AND NOT gates follow thekinetic segregation based model and a correct spacer must be selected tocreate a functional gate (FIG. 23B).

Example 10: Summary of Model of CAR Logic Gates Generated by KineticSegregation

Based on current understanding of the kinetic-segregation model and theexperimental data described herein, a summary of the model for a two-CARgate is presented in FIG. 24. The Figure shows a cell expressing twoCARs, each recognizing a different antigen. When either or both CARsrecognize a target antigen on a cell, a synapse forms and native CD45and CD148 are excluded from the synapse due to the bulk of theirectodomain. This sets the stage for T-cell activation. In the case thatthe target cell bears only one cognate antigen, the cognate CAR isligated and the cognate CAR segregates into the synapse. The unligatedCAR remains in solution on the T-cell membrane and can diffuse in andout of the synapse so that an area of high local concentration ofligated CAR with low concentration of unligated CAR forms. In this case,if the ligated CAR has an ITAM and the non-ligated CAR has ‘ligationoff” type inhibitory endodomain such as that of CD148, the amount ofnon-ligated CAR is sufficient to inhibit activation and the gate is off.In contrast, in this case, if the ligated CAR has an ITAM and thenon-ligated CAR has a ‘ligation on’ type inhibitory endodomain such asPTPN6, the amount of non-ligated CAR is insufficient to inhibit and thegate is on. When challenged by a target cell bearing both cognateantigens, both cognate CARs are ligated and form part of animmunological synapse. Importantly, if the CAR spacers are sufficientlysimilar, the CARs co-localize in the synapse but if the CAR spacers aresufficiently different the CARs segregate within the synapse. In thislatter case, areas of membrane form whereby high concentrations of oneCAR are present but the other CAR is absent. In this case sincesegregation is complete, even if the inhibitory endodomain is a‘ligation off’ type, the gate is on. In the former case, areas ofmembrane form with high concentrations of both CARs mixed together. Inthis case, since both endodomains are concentrated, even if theinhibitory endodomain is ‘ligation on’ type, the gate is off. Byselecting the correct combination of spacer and endodomain logic can beprogrammed into a CAR T-cell.

Based on our work above, we have established a series of design rules toallow generation of logic-gated CARs (illustrated in FIG. 31). Togenerate an “antigen A OR antigen B” gated CAR T-cell, anti-A and anti-BCARs must be generated such that (1) each CAR has a spacer which simplyallows antigen access and synapse formation such that the CAR functions,and (2) Each CAR has an activating endodomain; To generate an “antigen AAND NOT B” gated CAR T-cell, anti-A and anti-B CARs must be generatedsuch that (1) both CARs have spacers which do not cross-pair, but whichwill allow the CARs to co-segregate upon recognition of both cognateantigens on the target cell, (2) and one CAR has an activatingendodomain, while the other CAR has an endodomain which comprises orrecruits a weak phosphatase (e.g. PTPN6); (3) To generate an “antigen AAND antigen B” gated CAR T-cell, anti-A and anti-B CARs must begenerated such that (1) one CAR has a spacer sufficiently different fromthe other CAR such that both CARs will not co-segregate upon recognitionof both cognate antigens on the target cell, (2) one CAR has anactivating endodomain, while the other car has an endodomain whichcomprises of a potent phosphatase (e.g. that of CD45 or CD148). Thecorrect spacers to achieve the desired effect can be selected from a setof spacers with known size/shape etc as well as taking intoconsideration size/shape etc of the target antigen and the location ofthe cognate epitope on the target antigen.

SEQ ID No 41: SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVQLQESGPGLVKPSQTLSITCTVSGFSLASYNIHWVRQPPGKGLEWLGVIWAGGSTNYNSALMSRLTISKDNSKNQVFLKMSSLTAADTAVYYCAKRSDDYSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSENQMTQSPSSLSASVGDRVTMTCRASSSVSSSYLHWYQQKSGKAPKVWIYSTSNLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSGYPITFGQGTKVEIKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIASEQ ID No. 42: SFG.aCD19-CD8STK-CD28tmZ-2A-aGD2-HCH2CH3pvaa-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCAGGTGCAGCTGCAGGAGTCTGGCCCAGGCCTGGTGAAGCCCAGCCAGACCCTGAGCATCACCTGCACCGTGAGCGGCTTCAGCCTGGCCAGCTACAACATCCACTGGGTGCGGCAGCCCCCAGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGCTGGCGGCAGCACCAACTACAACAGCGCCCTGATGAGCCGGCTGACCATCAGCAAGGACAACAGCAAGAACCAGGTGTTCCTGAAGATGAGCAGCCTGACAGCCGCCGACACCGCCGTGTACTACTGCGCCAAGCGGAGCGACGACTACAGCTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGAACCAGATGACCCAGAGCCCCAGCAGCTTGAGCGCCAGCGTGGGCGACCGGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCAGCAGCTACCTGCACTGGTACCAGCAGAAGAGCGGCAAGGCCCCAAAGGTGTGGATCTACAGCACCAGCAACCTGGCCAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCGGCTACCCCATCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAASEQ ID No. 43: SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDVYYNPSLKNQLTISKDASRDQVFLKITNLDTADTATYYCVRRRATGTGFDYWGQGTTLTVSSGGGGSGGGGSGGGGSNIVMTQSHKFMSTSVGDRVSIACKASQDVGTAVAWYQQKPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYFCHQYNSYNTFGSGTRLELKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIASEQ ID No. 44: SFG.aCD19-CD8STK-CD28tmZ-2A-aCD5-HCH2CH3pvaa-dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCAGGTGACCCTGAAGGAGAGCGGTCCCGGCATCCTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCAGCTTCAGCGGCTTCAGCCTGAGCACCAGCGGCATGGGCGTGGGCTGGATTCGGCAGCCCAGCGGCAAGGGCCTGGAGTGGCTGGCCCACATCTGGTGGGACGACGACGTGTACTACAACCCCAGCCTGAAGAACCAGCTGACCATCAGCAAGGACGCCAGCCGGGACCAGGTGTTCCTGAAGATCACCAACCTGGACACCGCCGACACCGCCACCTACTACTGCGTGCGGCGCCGGGCCACCGGCACCGGCTTCGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGGTGGCGGTGGCAGCGGCGGCGGCGGAAGCGGAGGTGGTGGCAGCAACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGACCGGGTGAGCATCGCCTGCAAGGCCAGCCAGGACGTGGGCACCGCCGTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCCAAGCTGCTGATCTACTGGACCAGCACCCGGCACACCGGCGTGCCCGACCGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCACCAACGTGCAGAGCGAGGACCTGGCCGACTACTTCTGCCACCAGTACAACAGCTACAACACCTTCGGCAGCGGCACCCGGCTGGAGCTGAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAASEQ ID No. 45: SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRAEGRGSLLTCGDVEENPGPMETDTLLLWVLLLWVPGSTGQVKLQQSGGGLVKPGASLKLSCVTSGFTFRKFGMSWVRQTSDKRLEWVASISTGGYNTYYSDNVKGRFTISRENAKNTLYLQMSSLKSEDTALYYCTRGYSSTSYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPASLSVATGEKVTIRCMTSTDIDDDMNWYQQKPGEPPKFLISEGNTLRPGVPSRFSSSGTGTDFVFTIENTLSEDVGDYYCLQSFNVPLTFGDGTKLEIKRSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAVFGCIFGALVIVTVGGFIFWRKKRKDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIASEQ ID No. 46: SFG.aCD19-CD8STK-CD28tmZ-2A-aEGFRvIII-HCH2CH3pvaa- dCD148ATGAGCCTGCCCGTGACCGCCCTGCTGCTGCCCCTGGCCCTGCTGCTGCACGCCGCCAGACCAGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACCGGGTGACCATCAGCTGCAGAGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTCTGCCAGCAGGGCAACACCCTGCCCTACACCTTCGGAGGCGGCACCAAGCTGGAGATCACCAAGGCCGGAGGCGGAGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCGAGGTGAAGCTGCAGGAGTCTGGCCCAGGCCTGGTGGCCCCAAGCCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGGCAGCCCCCACGGAAGGGCCTGGAGTGGCTGGGCGTGATCTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTATGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCTCAGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCAGAGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCCGGCAGCACCGGCCAGGTGAAGCTGCAGCAGAGCGGCGGAGGCCTGGTGAAGCCCGGCGCCAGCCTGAAGCTGAGCTGCGTGACCAGCGGCTTCACCTTCCGGAAGTTCGGCATGAGCTGGGTGCGGCAGACCAGCGACAAGCGGCTGGAGTGGGTGGCCAGCATCAGCACCGGCGGCTACAACACCTACTACAGCGACAACGTGAAGGGCCGGTTCACCATCAGCCGGGAGAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAAGAGCGAGGACACCGCCCTGTACTACTGCACCCGGGGCTACAGCAGCACCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACAGTGAGCAGCGGCGGAGGAGGCAGTGGTGGGGGTGGATCTGGCGGAGGTGGCAGCGACATCGAGCTGACCCAGAGCCCCGCCAGCCTGAGCGTGGCCACCGGCGAGAAGGTGACCATCCGGTGCATGACCAGCACCGACATCGACGACGACATGAACTGGTACCAGCAGAAGCCCGGCGAGCCCCCAAAGTTCCTGATCAGCGAGGGCAACACCCTGCGGCCCGGCGTGCCCAGCCGGTTCAGCAGCAGCGGCACCGGCACCGACTTCGTGTTCACCATCGAGAACACCCTGAGCGAGGACGTGGGCGACTACTACTGCCTGCAGAGCTTCAACGTGCCCCTGACCTTCGGCGACGGCACCAAGCTGGAGATCAAGCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCCCTGCACAATCACTATACCCAGAAATCTCTGAGTCTGAGCCCAGGCAAGAAGGACCCCAAGGCGGTTTTTGGCTGTATCTTTGGTGCCCTGGTTATTGTGACTGTGGGAGGCTTCATCTTCTGGAGAAAGAAGAGGAAAGATGCAAAGAATAATGAAGTGTCCTTTTCTCAAATTAAACCTAAAAAATCTAAGTTAATCAGAGTGGAGAATTTTGAGGCCTACTTCAAGAAGCAGCAAGCTGACTCCAACTGTGGGTTCGCAGAGGAATACGAAGATCTGAAGCTTGTTGGAATTAGTCAACCTAAATATGCAGCAGAACTGGCTGAGAATAGAGGAAAGAATCGCTATAATAATGTTCTGCCCTATGATATTTCCCGTGTCAAACTTTCGGTCCAGACCCATTCAACGGATGACTACATCAATGCCAACTACATGCCTGGCTACCACTCCAAGAAAGATTTTATTGCCACACAAGGACCTTTACCGAACACTTTGAAAGATTTTTGGCGTATGGTTTGGGAGAAAAATGTATATGCCATCATTATGTTGACTAAATGTGTTGAACAGGGAAGAACCAAATGTGAGGAGTATTGGCCCTCCAAGCAGGCTCAGGACTATGGAGACATAACTGTGGCAATGACATCAGAAATTGTTCTTCCGGAATGGACCATCAGAGATTTCACAGTGAAAAATATCCAGACAAGTGAGAGTCACCCTCTGAGACAGTTCCATTTCACCTCCTGGCCAGACCACGGTGTTCCCGACACCACTGACCTGCTCATCAACTTCCGGTACCTCGTTCGTGACTACATGAAGCAGAGTCCTCCCGAATCGCCGATTCTGGTGCATTGCAGTGCTGGGGTCGGAAGGACGGGCACTTTCATTGCCATTGATCGTCTCATCTACCAGATAGAGAATGAGAACACCGTGGATGTGTATGGGATTGTGTATGACCTTCGAATGCATAGGCCTTTAATGGTGCAGACAGAGGACCAGTATGTTTTCCTCAATCAGTGTGTTTTGGATATTGTCAGATCCCAGAAAGACTCAAAAGTAGATCTTATCTACCAGAACACAACTGCAATGACAATCTATGAAAACCTTGCGCCCGTGACCACATTTGGAAAGACCAATGGTTACATCGCCTAA

Example 11: Design and Construction of APRIL Based CARs

APRIL in its natural form is a secreted type II protein. The use ofAPRIL as a BCMA binding domain for a CAR requires conversion of thistype II secreted protein to a type I membrane bound protein and for thisprotein to be stable and to retain binding to BCMA in this form. Togenerate candidate molecules, the extreme amino-terminus of APRIL wasdeleted to remove binding to proteoglycans. Next, a signal peptide wasadded to direct the nascent protein to the endoplasmic reticulum andhence the cell surface. Also, because the nature of spacer used canalter the function of a CAR, three different spacer domains were tested:an APRIL based CAR was generated comprising (i) a human IgG1 spaceraltered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) theIgG1 hinge alone (cartoon in FIG. 25 and amino acid sequences in FIG.26). These CARs were expressed in a bicistronic retroviral vector (FIG.27A) so that a marker protein—truncated CD34 could be co-expressed as aconvenient marker gene.

Example 12: Expression and Function of APRIL Based CARs

The aim of this study was to test whether the APRIL based CARs which hadbeen constructed were expressed on the cell surface and whether APRILhad folded to form the native protein. T-cells were transduced withthese different CAR constructs and stained using a commerciallyavailable anti-APRIL mAb, along with staining for the marker gene andanalysed by flow-cytometry. The results of this experiment are shown inFIG. 27B where APRIL binding is plotting against marker genefluorescence. These data show that in this format, the APRIL based CARsare expressed on the cell surface and APRIL folds sufficiently to berecognized by an anti-APRIL mAb.

Next, it was determined whether APRIL in this format could recognizeBCMA and TACl. Recombinant BCMA and TACl were generated as fusions withmouse IgG2a-Fc. These recombinant proteins were incubated with thetransduced T-cells. After this, the cells were washed and stained withan anti-mouse fluorophore conjugated antibody and an antibody to detectthe marker gene conjugated to a different fluorophore. The cells wereanalysed by flow cytometry and the results are presented in FIG. 27C.The different CARs were able to bind both BCMA and TACl. Surprisingly,the CARs were better able to bind BCMA than TACl. Also, surprisinglyCARs with a CD8 stalk or IgG1 hinge spacer were better able to bind BCMAand TACl than CAR with an Fc spacer.

Example 13: APRIL Based Chimeric Antigen Receptors are Active AgainstBCMA Expressing Cells

T-cells from normal donors were transduced with the different APRIL CARsand tested against SupT1 cells either wild-type, or engineered toexpress BCMA and TACI. Several different assays were used to determinefunction. A classical chromium release assay was performed. Here, thetarget cells (the SupT1 cells) were labelled with 51Cr and mixed witheffectors (the transduced T-cells) at different ratio. Lysis of targetcells was determined by counting 51Cr in the co-culture supernatant(FIG. 28A shows the cumulative data).

In addition, supernatant from T-cells cultured 1:1 with SupT1 cells wasassayed by ELISA for Interferon-gamma (FIG. 28B shows cumulative data).Measurement of T-cell expansion after one week of co-culture with SupT1cells was also performed (FIG. 28C). T-cells were counted byflow-cytometry calibrated with counting beads. These experimental datashow that APRIL based CARs can kill BCMA expressing targets. Further,these data show that CARs based on the CD8 stalk or IgG1 hinge performedbetter than the Fc-pvaa based CAR.

Example 14: Functional Analysis of the AND Gate in Primary Cells

PBMCs were isolated from blood and stimulated using PHA and IL-2. Twodays later the cells were transduced on retronectin coated plates withretro virus containing the CD19:CD33 AND gate construct. On day 5 theexpression level of the two CARs translated by the AND gate constructwas evaluated via flow cytometry and the cells were depleted of CD56+cells (predominantly NK cells). On day 6 the PBMCs were placed in aco-culture with target cells at a 1:2 effector to target cell ratio. Onday 8 the supernatant was collected and analysed for IFN-gamma secretionvia ELISA (FIG. 29).

These data demonstrate that the AND gate functions in primary cells.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, cell biology or related fields are intended to bewithin the scope of the following claims.

1. A T cell which co-expresses a first chimeric antigen receptor (CAR)and second CAR at the cell surface, each CAR comprising: (i) anantigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and(iv) an endodomain wherein the antigen binding domains of the first andsecond CARs bind to different antigens, and wherein each of the firstand second CARs is an activating CAR comprising an activatingendodomain, wherein the spacer of the first CAR has a different lengthand/or configuration from the spacer of the second CAR, such that eachCAR is tailored for recognition of its respective target antigen.
 2. A Tcell according to claim 1, wherein either the first CAR spacer or thesecond CAR spacer comprises a CD8 stalk and the other spacer comprisesthe hinge, CH2 and CH3 domain of IgG1.
 3. (canceled)
 4. A T cellaccording to claim 1, wherein one CAR binds CD19 and the other CAR bindsCD20.
 5. A T cell according to claim 1 which comprises more than twoCARs such that it is specifically stimulated by a cell, such as a targetcell, bearing a distinct pattern of more than two antigens.
 6. A nucleicacid sequence encoding both first and second chimeric antigen receptors(CARs) each CAR comprising: (i) an antigen-binding domain; (ii) a spacer(iii) a trans-membrane domain; and (iv) an endodomain wherein theantigen binding domains of the first and second CARs bind to differentantigens, and wherein each of the first and second CARs is an activatingCAR comprising an activating endodomain, wherein the spacer of the firstCAR has a different length and/or configuration from the spacer of thesecond CAR, such that each CAR is tailored for recognition of itsrespective target antigen.
 7. A nucleic acid sequence according to claim6, which has the following structure:AgB1-spacer1-TM1-endo1-coexpr-AbB2-spacer2-TM2-endo2 in which AgB1 is anucleic acid sequence encoding the antigen-binding domain of the firstCAR; spacer 1 is a nucleic acid sequence encoding the spacer of thefirst CAR; TM1 is a nucleic acid sequence encoding the transmembranedomain of the first CAR; endo 1 is a nucleic acid sequence encoding theendodomain of the first CAR; coexpr is a nucleic acid sequence enablingco-expression of both CARs AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CAR; whichnucleic acid sequence, when expressed in a T cell, encodes a polypeptidewhich is cleaved at the cleavage site such that the first and secondCARs are co-expressed at the T cell surface.
 8. (canceled)
 9. A nucleicacid sequence according to claim 7, wherein alternative codons are usedin regions of sequence encoding the same or similar amino acidsequences, in order to avoid homologous recombination.
 10. A kit whichcomprises (i) a first nucleic acid sequence encoding a first chimericantigen receptor (CAR), which nucleic acid sequence has the followingstructure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR; TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and (ii)a second nucleic acid sequence encoding a second chimeric antigenreceptor (CAR), which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigen.
 11. A kit comprising: a first vectorwhich comprises the first nucleic acid sequence nucleic acid sequence asdefined in claim 10 encoding a first chimeric antigen receptor (CAR),which nucleic acid sequence has the following structure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR; TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and asecond vector which comprises a second nucleic acid sequence encoding asecond CAR, which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigens.
 12. A kit according to claim 11,wherein the vectors are integrating viral vectors or transposons.
 13. Avector comprising a nucleic acid sequence according to claim
 6. 14. Aretroviral vector or a lentiviral vector or a transposon according toclaim
 13. 15. A method for making a T cell according to claim 1, whichcomprises the step of introducing into a T cell: (i) a nucleic acidsequence or a vector comprising a nucleic acid sequence encoding bothfirst and second chimeric antigen receptors (CARs), each CAR comprising:(a) an antigen-binding domain; (b) a spacer (c) a trans-membrane domain;and (d) an endodomain wherein the antigen binding domains of the firstand second CARs bind to different antigens, and wherein each of thefirst and second CARs is an activating CAR comprising an activatingendodomain, wherein the spacer of the first CAR has a different lengthand/or configuration from the spacer of the second CAR, such that eachCAR is tailored for recognition of its respective target antigen; (ii) akit comprising (a) a first nucleic acid sequence encoding a firstchimeric antigen receptor (CAR), which nucleic acid sequence has thefollowing structure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR; TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and (b)a second nucleic acid sequence encoding a second chimeric antigenreceptor (CAR), which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigen; or (iii) a kit comprising: (a) a firstvector which comprises the first nucleic acid sequence encoding a firstchimeric antigen receptor (CAR), which nucleic acid sequence has thefollowing structure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR: TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and (b)a second vector which comprises a second nucleic acid sequence encodinga second CAR, which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigens.
 16. A method according to claim 15,wherein the T cell is from a sample isolated from a subject.
 17. Apharmaceutical composition comprising a plurality of T cells accordingto claim
 1. 18. A method for treating and/or preventing a disease, whichcomprises the step of administering a pharmaceutical compositionaccording to claim 17 to a subject.
 19. A method according to claim 18,which comprises the following steps: (i) isolation of a Tcell-containing sample from a subject; (ii) transduction or transfectionof the T cells with: (a) a nucleic acid sequence or a vector comprisinga nucleic acid sequence encoding both first and second chimeric antigenreceptors (CARs), each CAR comprising: (1) an antigen-binding domain;(2) a spacer (3) a trans-membrane domain; and (4) an endodomain whereinthe antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigen; (b) a kit comprising (1) a firstnucleic acid sequence encoding a first chimeric antigen receptor (CAR),which nucleic acid sequence has the following structure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR: TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and (2)a second nucleic acid sequence encoding a second chimeric antigenreceptor (CAR), which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigen; or (c) a kit comprising: (1) a firstvector which comprises the first nucleic acid sequence encoding a firstchimeric antigen receptor (CAR), which nucleic acid sequence has thefollowing structure:AgB1-spacer1-TM1-endo1 in which AgB1 is a nucleic acid sequence encodingthe antigen-binding domain of the first CAR; spacer 1 is a nucleic acidsequence encoding the spacer of the first CAR: TM1 is a nucleic acidsequence encoding the transmembrane domain of the first CAR; endo 1 is anucleic acid sequence encoding the endodomain of the first CAR; and (2)a second vector which comprises a second nucleic acid sequence encodinga second CAR, which nucleic acid sequence has the following structure:AgB2-spacer2-TM2-endo2 AgB2 is a nucleic acid sequence encoding theantigen-binding domain of the second CAR; spacer 2 is a nucleic acidsequence encoding the spacer of the second CAR; TM2 is a nucleic acidsequence encoding the transmembrane domain of the second CAR; endo 2 isa nucleic acid sequence encoding the endodomain of the second CARwherein the antigen binding domains of the first and second CARs bind todifferent antigens, and wherein each of the first and second CARs is anactivating CAR comprising an activating endodomain, wherein the spacerof the first CAR has a different length and/or configuration from thespacer of the second CAR, such that each CAR is tailored for recognitionof its respective target antigens; and (iii) administering the T cellsfrom (ii) to a the subject.
 20. A method according to claim 19, whereinthe disease is a cancer. 21-26. (canceled)
 27. A natural killer (NK)cell which co-expresses a first chimeric antigen receptor (CAR) andsecond CAR at the cell surface, each CAR comprising: (i) anantigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and(iv) an endodomain wherein the antigen binding domains of the first andsecond CARs bind to different antigens, and wherein each of the firstand second CARs is an activating CAR comprising an activatingendodomain, wherein the spacer of the first CAR has a different lengthand/or configuration from the spacer of the second CAR, such that eachCAR is tailored for recognition of its respective target antigen.
 28. Acell composition comprising: (i) T cells according to claim 1, and/or(ii) NK cells which co-express a first chimeric antigen receptor (CAR)and second CAR at the cell surface, each CAR comprising: (i) anantigen-binding domain; (ii) a spacer (iii) a trans-membrane domain; and(iv) an endodomain wherein the antigen binding domains of the first andsecond CARs bind to different antigens, and wherein each of the firstand second CARs is an activating CAR comprising an activatingendodomain, wherein the spacer of the first CAR has a different lengthand/or configuration from the spacer of the second CAR, such that eachCAR is tailored for recognition of its respective target antigen made bytransducing a blood-sample ex vivo with a nucleic acid encoding thefirst and second CARs.